Consensus/ancestral immunogens

ABSTRACT

The present invention relates, in general, to an immunogen and, in particular, to an immunogen for inducing antibodies that neutralizes a wide spectrum of HIV primary isolates and/or to an immunogen that induces a T cell immune response. The invention also relates to a method of inducing anti-HIV antibodies, and/or to a method of inducing a T cell immune response, using such an immunogen. The invention further relates to nucleic acid sequences encoding the present immunogens.

This application is a Continuation of U.S. application Ser. No.13/137,517, filed Aug. 23, 2011, which is a Continuation of U.S.application Ser. No. 10/572,638, filed Dec. 22, 2006, which is aNational Stage Application under 35 U.S.C. section 371 ofPCT/US2004/030397, filed Sep. 17, 2004, which claims priority from U.S.Provisional Application No. 60/503,460, filed Sep. 17, 2003, and U.S.Provisional Application No. 60/604,722, filed Aug. 27, 2004, the entirecontents of which are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Jun. 28, 2018, is named2933311-030-US7_SL.txt and is 1,108,293 bytes in size.

TECHNICAL FIELD

The present invention relates, in general, to an immunogen and, inparticular, to an immunogen for inducing antibodies that neutralize awide spectrum of HIV primary isolates and/or to an immunogen thatinduces a T cell immune response. The invention also relates to a methodof inducing anti-HIV antibodies, and/or to a method of inducing a T cellimmune response, using such an immunogen. The invention further relatesto nucleic acid sequences encoding the present immunogens.

BACKGROUND

The high level of genetic variability of HIV-1 has presented a majorhurdle for AIDS vaccine development. Genetic differences among HIV-1groups M, N, and O are extensive, ranging from 30% to 50% in gag and envgenes, respectively (Gurtler et al, J. Virol. 68:1581-1585 (1994),Vanden Haesevelde et al, J. Virol. 68:1586-1596 (1994), Simon et al,Nat. Med. 4:1032-1037 (1998), Kuiken et al, Human retroviruses and AIDS2000: a compilation and analysis of nucleic acid and amino acidsequences (Theoretical Biology and Biophysics Group, Los Alamos NationalLaboratory, Los Alamos, N. Mex.)). Viruses within group M are furtherclassified into nine genetically distinct subtypes (A-D, F-H, J and K)(Kuiken et al, Human retroviruses and AIDS 2000: a compilation andanalysis of nucleic acid and amino acid sequences (Theoretical Biologyand Biophysics Group, Los Alamos National Laboratory, Los Alamos, NewMex., Robertson et al, Science 288:55-56 (2000), Robertson et al, Humanretroviruses and AIDS 1999: a compilation and analysis of nucleic acidand amino acid sequences, eds. Kuiken et al (Theoretical Biology andBiophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex.),pp. 492-505 (2000)). With the genetic variation as high as 30% in envgenes among HIV-1 subtypes, it has been difficult to consistently elicitcross-subtype T and B cell immune responses against all HIV-1 subtypes.HIV-1 also frequently recombines among different subtypes to createcirculating recombinant forms (CRFs) (Robertson et al, Science 288:55-56(2000), Robertson et al, Human retroviruses and AIDS 1999: a compilationand analysis of nucleic acid and amino acid sequences, eds. Kuiken et al(Theoretical Biology and Biophysics Group, Los Alamos NationalLaboratory, Los Alamos, N. Mex.), pp. 492-505 (2000), Carr et al, Humanretroviruses and AIDS 1998: a compilation and analysis of nucleic acidand amino acid sequences, eds. Korber et al (Theoretical Biology andBiophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex.),pp. III-10-III-19 (1998)). Over 20% of HIV-1 isolates are recombinant ingeographic areas where multiple subtypes are common (Robertson et al,Nature 374:124-126 (1995), Cornelissen et al, J. virol. 70:8209-8212(1996), Dowling et al, AIDS 16:1809-1820 (2002)), and high prevalencerates of recombinant viruses may further complicate the design ofexperimental HIV-1 immunogens.

To overcome these challenges in AIDS vaccine development, three computermodels (consensus, ancestor and center of the tree) have been used togenerate centralized HIV-1 genes to (Gaschen et al, Science296:2354-2360 (2002), Gap et al, Science 299:1517-1518 (2003), Nickle etal, Science 299:1515-1517 (2003), Novitsky et al, J. Virol. 76:5435-5451(2002), Ellenberger et al, Virology 302:155-163 (2002), Korber et al,Science 288:1789-1796 (2000)). The biology of HIV gives rise tostar-like phylogenies, and as a consequence of this, the three kinds ofsequences differ from each other by 2-5% (Gao et al, Science299:1517-1518 (2003)). Any of the three centralized gene strategies willreduce the protein distances between immunogens and field virus strains.Consensus sequences minimize the degree of sequence dissimilaritybetween a vaccine strain and contemporary circulating viruses bycreating artificial sequences based on the most common amino acid ineach position in an alignment (Gaschen et al, Science 296:2354-2360(2002)). Ancestral sequences are similar to consensus sequences but aregenerated using maximum-likelihood phylogenetic analysis methods(Gaschen et al, Science 296:2354-2360 (2002), Nickle et al, Science299:1515-1517 (2003)). In doing so, this method recreates thehypothetical ancestral genes of the analyzed current wild-type sequences(FIG. 26). Nickle et al proposed another method to generate centralizedHIV-1 sequences, center of the tree (COT), that is similar to ancestralsequences but less influenced by outliers (Science 299:1515-1517(2003)).

The present invention results, at least in part, from the results ofstudies designed to determine if centralized immunogens can induce bothT and B cell immune responses in animals. These studies involved thegeneration of an artificial group M consensus env gene (CON6), andconstruction of DNA plasmids and recombinant vaccinia viruses to expressCON6 envelopes as soluble gp120 and gp140CF proteins. The resultsdemonstrate that CON6 Env proteins are biologically functional, possesslinear, conformational and glycan-dependent epitopes of wild-type HIV-1,and induce cytokine-producing T cells that recognize T cell epitopes ofboth HIV subtypes B and C. Importantly, CON6 gp120 and gp140CF proteinsinduce antibodies that neutralize subsets of subtype B and C HIV-1primary isolates.

The iterative nature of study of the centralized HIV-1 gene approach isderived from the rapidly expanding evolution of HIV-1 sequences, and thefact that sequences collected in the HIV sequence database (that is, theLos Alamos National Database) are continually being updated with newsequences each year. The CON6 gp120 envelope gene derives from Year 1999Los Alamos National Database sequences, and Con-S derives from Year 2000Los Alamos National Database sequences. In addition, CON6 has Chinesesubtype C V1, V2, V4, and V5 Env sequences, while Con-S has all group Mconsensus Env constant and variable regions, that have been shortened tominimal-length variable loops. Codon-optimized genes for a series ofYear 2003 group M and subtype consensus sequences have been designed, ashave a corresponding series of wild-type HIV-1 Env genes for comparison,for use in inducing broadly reactive T and B cell responses to HIV-1primary isolates.

SUMMARY OF THE INVENTION

The present invention relates to an immunogen for inducing antibodiesthat neutralize a wide spectrum of HIV primary isolates and/or to animmunogen that induces a T cell immune response, and to nucleic acidsequences encoding same. The invention also relates to a method ofinducing anti-HIV antibodies, and/or to a method of inducing a T cellimmune response, using such an immunogen.

Objects and advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D: Generation and expression of the group M consensus env gene(CON6). The complete amino acid sequence of CON6 gp160 is shown. (FIG.1A) (SEQ ID NO: 1) The five regions from the wild-type CRF08_BC(98CN006) env gene are indicated by underlined letters. Variable regionsare indicated by brackets above the sequences. Potential N-likedglycosylation sites are highlighted with bold-faced letters. (FIG. 1B)Constructs of CON6 gp120 and gp140CF. CON6 gp120 and gp140CF plasmidswere engineered by introducing a stop codon after the gp120 cleavagesite or before the transmembrane domain, respectively. The gp120/gp41cleavage site and fusion domain of gp41 were deleted in the gp140CFprotein. (FIG. 1C) Expression of CON6 gp120 and gp140CF. CON6 gp120 andgp140CF were purified from the cell culture supernatants of rVV-infected293T cells with galanthus Nivalis argarose lectin columns. Both gp120and gp140CF were separated on a 10% SDS-polyarylamide gel and stainedwith Commassie blue. (FIG. 1D.) (SEQ ID NO: 2) CON6 env gene optimizedbased on codon usage for highly expressed human genes.

FIGS. 2A-2E. Binding of CON6 gp120 gp140 CF to soluble CD4 (sCD4) andanti-Env mAbs. (FIGS. 2A-2B) Each of the indicated mabs and sCD4 wascovalently immobilized to a CM5 sensor chip (BIAcore) and CON6 gp120(FIG. 2A) or gp140CF (FIG. 2B) (100 μg/ml and 300 μg/ml, respectively)were injected over each surface. Both gp120 and gp140CF proteins reactedwith each anti-gp120 mabs tested except for 17b mab, which showednegligible binding to both CON6 gp120 and gp140CF. To determineinduction of 17b mab binding to CON6 gp120 and gp140CF, CON6 gp120 (FIG.2C) or gp140CF (FIG. 2D) proteins were captured (400-580 RU) onindividual flow cells immobilized with sCD4 or mabs A32 or T8. Followingstabilization of each of the surface, mAb 17b was injected and flowedover each of the immobilized flow cells. Overlay of curves show that thebinding of mab 17b to CON6 Env proteins was markedly enhanced on bothsCD4 and mab A32 surfaces but not on the T8 surface (FIGS. 2C-2D). Todetermine binding of CON6 gp120 and gp140CF to human mabs in ELISA,stock solutions of 20: g/ml of mabs 447, F39F, A32, IgG1b12 and 2F5 onCON6 gp120 and gp140CF were tittered (FIG. 2E). Mabs 447 (V3), F39F (V3)A32 (gp120) and IgG1b12 (CD4 binding site) each bound to both CON6 gp120and 140 well, while 2F5 (anti-gp41 ELDKWAS) (SEQ ID NO: 321) only boundgp140CF. The concentration at endpoint titer on gp120 for mab 447 andF39F binding was <0.003 μg/ml and 0.006 μg/ml, respectively; for mab A32was <0.125 μg/ml; for IgG1b12 was <0.002 μg/ml; and for 2F5 was 0.016μg/ml.

FIGS. 3A and 3B. Infectivity and coreceptor usage of CON6 envelope.(FIG. 3A) CON6 and control env plasmids were cotransfected withHIV-1/SG3Δenv backbone into human 293T cells to generateEnv-pseudovirions. Equal amounts of each pseudovirion (5 ng p24) wereused to infect JC53-BL cells. The infectivity was determined by countingthe number of blue cells (infectious units, IU) per microgram of p24 ofpseudovirons (IU/μg p24) after staining the infected cells for β-galexpression. (FIG. 3B) Coreceptor usage of the CON6 env gene wasdetermined on JC53BL cells treated with AMD3100 and/or TAK-799 for 1 hr(37° C.) then infected with equal amounts of p24 (5 ng) of eachEnv-pseudovirion. Infectivity in the control group (no blocking agent)was set as 100%. Blocking efficiency was expressed as the percentage ofIU from blocking experiments compared to those from control cultureswithout blocking agents. Data shown are mean±SD.

FIG. 4. Western blot analysis of multiple subtype Env proteins againstmultiple subtype antisera. Equal amount of Env proteins (100 ng) wereseparated on 10% SDS-polyacrylamide gels. Following electrophoresis,proteins were transferred to Hybond ECL nitrocellulose membranes andreacted with sera from HIV-1 infected patients (1:1,000) or guinea pigsimmunized with CON6 gp120 DNA prime, rVV boost (1:1,000). Protein-boundantibody was probed with fluorescent-labeled secondary antibodies andthe images scanned and recorded on an infrared imager Odyssey (Li-Cor,Lincoln, Nebr.). Subtypes are indicated by single-letters after Envprotein and serum IDs. Four to six sera were tested for each subtype,and reaction patterns were similar among all sera from the same subtype.One representative result for each subtype serum is shown.

FIG. 5. T cell immune responses induced by CON6 Env immunogens in mice.Splenocytes were isolated from individual immunized mice (5 mice/group).After splenocytes were stimulated in vitro with overlapping Env peptidepools of CON6 (black column), subtype B (hatched column), subtype C(white column), and medium (no peptide; gray column), INF-γ producingcells were determined by the ELISPOT assay. T cell IFN-γ responsesinduced by either CON6 gp120 or gp140CF were compared to those inducedby subtype specific Env immunogens (JRFL and 96ZM651). Total responsesfor each envelope peptide pool are expressed as SFCs per millionsplenocytes. The values for each column are the mean±SEM(of IFN-γ SFCs(n=5 mice/group).

FIGS. 6A-6E. Construction of codon usage optimized subtype C ancestraland consensus envelope genes (FIGS. 6A and 6B, respectively) (SEQ ID NOS3-4). Ancestral and consensus amino acid sequences (FIGS. 6C and 6D,respectively) (SEQ ID NOS 5-6) were transcribed to mirror the codonusage of highly expressed human genes. Paired oligonucleotides (80-mers)overlapping by 20 bp were designed to contain 5′ invariant sequencesincluding the restriction enzyme sites EcoRI, BbsI, Bam HI and BsmBI.BbsI and BsmBI are Type II restriction enzymes that cleave outside oftheir recognition sequences. Paired oligomers were linked individuallyusing PCR and primers complimentary to the 18 bp invariant sequences ina stepwise fashion, yielding 140 bp PCR products. These were subclonedinto pGEM-T and sequenced to confirm the absence of inadvertantmutations/deletions. Four individual pGEM-T subclones containing theproper inserts were digested and ligated together into pcDNA3.1.Multi-fragment ligations occurred repeatly amongst groups of fragmentsin a stepwise manner from the 5′ to the 3′ end of the gene until theentire gene was reconstructed in pcDNA3.1. (See schematic in FIG. 6E.)

FIG. 7. JC53-BL cells are a derivative of HeLa cells that express highlevels of CD4 and the HIV-1 coreceptors CCR5 and CXCR4. They alsocontain the reporter cassettes of luciferase and β-galactosidase thatare each expressed from an HIV-1 LTR. Expression of the reporter genesis dependent on production of HIV-1 Tat. Briefly, cells are seeded into24 or 96-well plates, incubated at 37° C. for 24 hours and treated withDEAE-Dextran at 37° C. for 30 minutes. Virus is serially diluted in 1%DMEM, added to the cells incubating in DEAE-Dextran, and allowed toincubate for 3 hours at 37° C. after which an additional cell media isadded to each well. Following a final 48-hour incubation at 37° C.,cells are either fixed, stained using X-Gal to visualize β-galactosidaseexpressing blue foci or frozen-thawed three times to measure luciferaseactivity.

FIG. 8. Sequence alignment of subtype C ancestral and consensus envgenes. Alignment of the subtype C ancestral (bottom line) (SEQ ID NO: 8)and consensus (top line) (SEQ ID NO: 7) env sequences showing a 95.5%sequence homology; amino acid sequence differences are indicated. Onenoted difference is the addition of a glycosylation site in the Cancestral env gene at the base of the V1 loop. A plus sign indicates awithin-class difference of amino acid at the indicated position; a barindicates a change in the class of amino acid. Potential N-glycosylationsites are marked in blue. The position of truncation for the gp140 geneis also shown.

FIG. 9. Expression of subtype C ancestral and consensus envelopes in293T cells. Plasmids containing codon-optimized gp160, gp140, or gp120subtype C ancestral and consensus genes were transfected into 293Tcells, and protein expression was examined by Western Blot analysis ofcell lysates. 48-hours post-transfection, cell lysates were collected,total protein content determined by the BCA protein assay, and 2 μg oftotal protein was loaded per lane on a 4-20% SDS-PAGE gel. Proteins weretransferred to a PVDF membrane and probed with HIV-1 plasma from asubtype C infected patient.

FIGS. 10A and 10B. FIG. 10A. Trans complementation of env-deficientHIV-1 with codon-optimized subtype C ancestral and consensus gp160 andgp140. Plasmids containing codon-optimized, subtype C ancestral orconsensus gp160 or gp140 genes were co-transfected into 293T cells withan HIV-1/SG3Aenv provirus. 48 hours post-transfection cell supernatantscontaining pseudotyped virus were harvested, clarified bycentrifugation, filtered through at 0.2 μM filter, and pelleted througha 20% sucrose cushion. Quantification of p24 in each virus pellet wasdetermined using the Coulter HIV-1 p24 antigen assay; 25 ng of p24 wasloaded per lane on a 4-20% SDS-PAGE gel for particles containing acodon-optimized envelope. 250 ng of p24 was loaded per lane forparticles generated by co-transfection of a rev-dependent wild-typesubtype C 96ZAM651env gene. Differences in the amount of p24 loaded perlane were necessary to ensure visualization of the rev-dependentenvelopes by Western Blot. Proteins were transferred to a PVDF membraneand probed with pooled plasma from HIV-1 subtype B and subtype Cinfected individuals. FIG. 10B. Infectivity of virus particlescontaining subtype C ancestral and consensus envelope glycoproteins.Infectivity of pseudotyped virus containing ancestral or consensus gp160or gp140 envelope was determined using the JC53-BL assay. Sucrosecushion purified virus particles were assayed by the Coulter p24 antigenassay, and 5-fold serial dilutions of each pellet were incubated withDEAE-Dextran treated JC53-BL cells. Following a 48-hour incubationperiod, cells were fixed and stained to visualize β-galactosidaseexpressing cells. Infectivity is represented as infectious units per ngof p24 to normalize for differences in the concentration of the inputpseudovirions.

FIG. 11. Co-receptor usage of subtype C ancestral and consensusenvelopes. Pseudotyped particles containing ancestral or consensusenvelope were incubated with DEAE-Dextran treated JC53-BL cells in thepresence of AMD3100 (a specific inhibitor of CXCR4), TAK779 (a specificinhibitor of CCR5), or AMD3000+TAK779 to determine co-receptor usage.NL4.3, an isolate known to utilize CXCR4, and YU-2, a known CCR5-usingisolate, were included as controls.

FIGS. 12A-12C. Neutralization sensitivity of subtype C ancestral andconsensus envelope glycoproteins. Equivalent amounts of pseudovirionscontaining the ancestral, consensus or 96ZAM651 gp160 envelopes (1,500infectious units) were preincubated with a panel of plasma samples fromHIV-1 subtype C infected patients and then added to the JC53-BL cellmonolayer in 96-well plates. Plates were cultured for two days andluciferase activity was measured as an indicator of viral infectivity.Virus infectivity is calculated by dividing the luciferase units (LU)produced at each concentration of antibody by the LU produced by thecontrol infection. The mean 50% inhibitory concentration (IC₅₀) and theactual % neutralization at each antibody dilution are then calculatedfor each virus. The results of all luciferase experiments are confirmedby direct counting of blue foci in parallel infections.

FIGS. 13A-13F. Protein expression of consensus subtype C Gag (FIG. 13A)and Nef (FIG. 13B) following transfection into 293T cells. Consensussubtype C Gag and Nef amino acid sequences are set forth in FIGS. 13Cand 13D (SEQ ID NOS: 9-10), respectively, and encoding sequences are setforth in FIGS. 13E and 13F (SEQ ID NOS: 11-12), respectively.

FIGS. 14A-14C. FIGS. 14A and 14B show the Con-S Env amino acid sequenceand encoding sequence, respectively (SEQ ID NOS: 13-14). FIG. 14C showsexpression of Group M consensus Con-S Env proteins using an in vitrotranscription and translation system.

FIGS. 15A and 15B. Expression of Con-S env gene in mammalian cells.(FIG. 15A—cell lysate, FIG. 15B—supernatant.)

FIGS. 16A and 16B. Infectivity (FIG. 16A) and coreceptor usage (FIG.16B) of CON6 and Con-S env genes.

FIGS. 17A-17C. Env protein incorporation in CON6 and Con-SEnv-pseudovirions. (FIG. 17A—lysate, FIG. 17B—supernatant, FIG. 17Cpellet.)

FIGS. 18A-18D. FIGS. 18A and 18B show subtype A consensus Env amino acidsequence and nucleic acid sequence encoding same, respectively (SEQ IDNOS: 15-16). FIGS. 18C and 18D show expression of A.con env gene inmammalian cells (FIG. 18C—cell lysate, FIG. 18D—supernatant).

FIGS. 19A-19H. M.con.gag (FIG. 19A) (SEQ ID NO: 17), M.con.pol (FIG.19B) (SEQ ID NO: 18), M.con.nef (FIG. 19C) (SEQ ID NO: 19) and C.con.pol(FIG. 19D) (SEQ ID NO: 20) nucleic acid sequences and correspondingencoded amino acid sequences (FIGS. 19E-19H, respectively) (SEQ ID NOS:21-24).

FIGS. 20A-20D. Subtype B consensus gag (FIG. 20A) (SEQ ID NO: 25) andenv (FIG. 20B) (SEQ ID NO: 26) genes. Corresponding amino acid sequencesare shown in FIGS. 20C and 20D (SEQ ID NOS: 28-29).

FIG. 21. Expression of subtype B consensus env and gag genes in 293Tcells. Plasmids containing codon-optimized subtype B consensus gp160,gp140, and gag genes were transfected into 293T cells, and proteinexpression was examined by Western Blot analysis of cell lysates.48-hours post-transfection, cell lysates were collected, total proteincontent determined by the BCA protein assay, and 2 μg of total proteinwas loaded per lane on a 4-20% SDS-PAGE gel. Proteins were transferredto a PVDF membrane and probed with serum from an HIV-1 subtype Binfected individual.

FIG. 22. Co-receptor usage of subtype B consensus envelopes. Pseudotypedparticles containing the subtype B consensus gp160 Env were incubatedwith DEAE-Dextran treated JC53-BL cells in the presence of AMD3100 (aspecific inhibitor of CXCR4), TAK779 (a specific inhibitor of CCR5), andAMD3000+TAK779 to determine co-receptor usage. NL4.3, an isolate knownto utilize CXCR4 and YU-2, a known CCR5-using isolate, were included ascontrols.

FIGS. 23A and 23B. Trans complementation of env-deficient HIV-1 withcodon-optimized subtype B consensus gp160 and gp140 genes. Plasmidscontaining codon-optimized, subtype B consensus gp160 or gp140 geneswere co-transfected into 293T cells with an HIV-1/SG3Δenv provirus.48-hours post-transfection cell supernatants containing pseudotypedvirus were harvested, clarified in a tabletop centrifuge, filteredthrough a 0.2 μM filter, and pellet through a 20% sucrose cushion.Quantification of p24 in each virus pellet was determined using theCoulter HIV-1 p24 antigen assay; 25 ng of p24 was loaded per lane on a4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane andprobed with anti-HIV-1 antibodies from infected HIV-1 subtype B patientserum. Trans complementation with a rev-dependent NL4.3 env was includedfor control. FIG. 23B. Infectivity of virus particles containing thesubtype B concensus envelope. Infectivitiy of pseudotyped viruscontaining consensus B gp160 or gp140 was determined using the JC53-BLassay. Sucrose cushion purified virus particles were assayed by theCoulter p24 antigen assay, and 5-fold serial dilutions of each pelletwere incubated with DEAE-Dextran treated JC53-BL cells. Following a48-hour incubation period, cells were fixed and stained to visualizeβ-galactosidase expressing cells. Infectivity is expressed as infectiousunits per ng of p24.

FIGS. 24A-24D. Neutralization sensitivity of virions containing subtypeB consensus gp160 envelope. Equivalent amounts of pseudovirionscontaining the subtype B consensus or NL4.3 Env (gp160) (1,500infectious units) were preincubated with three different monoclonalneutralizing antibodies and a panel of plasma samples from HIV-1 wubtypeB infected individuals, and then added to the JC53-BL cell monolayer in96-well plates. Plates were cultured for two days and luciferaseactivity was measured as an indicator of viral infectivity. Virusinfectivity was calculated by dividing the luciferase units (LU)produced at each concentration of antibody by the LU produced by thecontrol infection. The mean 50% inhibitory concentration (IC₅₀) and theactual % neutralization at each antibody dilution were then calculatedfor each virus. The results of all luciferase experiments were confirmedby direct counting of blue foci in parallel infections. FIG. 24A.Neutralization of Pseudovirions containing Subtype B consensus Env(gp160). FIG. 24B. Neutralization of Pseudovirions containing NL4.3 Env(gp160).

FIG. 24C. Neutralization of Pseudovirions containing Subtype B consensusEnv (gp160). FIG. 24D. Neutralization of Pseudovirions containing NL4.3Env (gp160).

FIGS. 25A and 25B. FIG. 25A. Density and p24 analysis of sucrosegradient fractions. 0.5 ml fractions were collected from a 20-60%sucrose gradient. Fraction number 1 represents the most dense fractiontaken from the bottom of the gradient tube. Density was measured with arefractometer and the amount of p24 in each fraction was determined bythe Coulter p24 antigen assay. Fractions 6-9, 10-15, 16-21, and 22-25were pooled together and analyzed by Western Blot. As expected, virionssedimented at a density of 1.16-1.18 g/ml.

FIG. 25B. VLP production by co-transfection of subtype B consensus gagand env genes. 293T cells were co-transfected with subtype B consensusgag and env genes. Cell supernatants were harvested 48-hourspost-transfection, clarified through at 20% sucrose cushion, and furtherpurified through a 20-60% sucrose gradient. Select fractions from thegradient were pooled, added to 20 ml of PBS, and centrifuged overnightat 100,000×g. Resuspended pellets were loaded onto a 4-20% SDS-PAGE gel,proteins were transferred to a PVDF membrane, and probed with plasmafrom an HIV-1 subtype B infected individual.

FIGS. 26A and 26B. FIG. 26A. 2000 Con-S 140CFI.ENV (SEQ ID NO: 30). FIG.26B. Codon-optimized Year 2000 Con-S 140CFI.seq (SEQ ID NO: 31).

FIG. 27. Individual C57BL/6 mouse T cell responses to HIV-1 envelopepeptides. Comparative immunogenicity of CON6 gp140CFI and Con-S gp140CFIin C57BL/C mice. Mice were immunized with either HIV5305 (Subtype A),2801 (Subtype B), CON6 or Con-S Envelope genes in DNA prime, rVV boostregimens, 5 mice per group. Spleen cells were assayed for IFN-γspot-forming cells 10 days after rVV boost, using mixtures ofoverlapping peptides from Envs of HIV-1 UG37(A), MN(B), Ch19(C), 89.6(B)SF162(B) or no peptide negative control.

FIGS. 28A-28C. FIG. 28A. Con-B 2003 Env. pep (841 a.a.) (SEQ ID NO: 32).Amino acid sequence underlined is the fusion domain that is deleted in140CF design and the “W” underlined is the last amino acid at theC-terminus, all amino acids after the “W” are deleted in the 140CFdesign. FIG. 28B. Con-B-140CF.pep (632 a.a.) (SEQ ID NO: 33). Aminoacids in bold identify the junction of the deleted fusion cleavage site.FIG. 28C. Codon-optimized Con-B 140CF.seq (1927 nt.) (SEQ ID NO: 34).

FIGS. 29A-29C. FIG. 29A. CON_OF_CONS-2003 (829 a.a.) (SEQ ID NO: 35).Amino acid sequence underlined is the fusion domain that is deleted in140CF design and the “W” underlined is the last amino acid at theC-terminus, all amino acids after the “W” are deleted in the 140CFdesign. FIG. 29B. ConS-2003 140CF.pep (620 a.a.) (SEQ ID NO: 36). Aminoacids in bold identify the junction of the deleted fusion cleavage site.FIG. 29C. CODON-OPTIMIZED ConS-2003 140CF.seq (1891 nt.) (SEQ ID NO:37).

FIGS. 30A-30C. FIG. 30A. CONSENSUS_A1-2003 (845 a.a.) (SEQ ID NO: 38).Amino acid sequence underlined is the fusion domain that is deleted in140CF design and the “W” underlined is the last amino acid at theC-terminus, all amino acids after the “W” are deleted in the 140CFdesign. FIG. 30B. Con-A1-2003 140CF.pep (629 a.a.) (SEQ ID NO: 39).Amino acids in bold identify the junction of the deleted fusion cleavagesite. FIG. 30C. CODON-OPTIMIZED Con-A1-2003.seq (SEQ ID NO: 40).

FIGS. 31A-31C. FIG. 31A. CONSENSUS_C-2003 (835 a.a.) (SEQ ID NO: 41).Amino acid sequence underlined is the fusion domain that is deleted in140CF design and the “W” underlined is the last amino acid at theC-terminus, all amino acids after the “W” are deleted in the 140CFdesign. FIG. 31B. Con-C 2003 140CF.pep (619 a.a.) (SEQ ID NO: 42). Aminoacids in bold identify the junction of the deleted fusion cleavage site.FIG. 31C. CODON-OPTIMIZED Con-C-2003 (140 CF (1,888 nt.) (SEQ ID NO:43).

FIGS. 32A-32C. FIG. 32A. CONSENSUS_G-2003 (842 a.a.) (SEQ ID NO: 44).Amino acid sequence underlined is the fusion domain that is deleted in140CF design and the “W” underlined is the last amino acid at theC-terminus, all amino acids after the “W” are deleted in the 140CFdesign. FIG. 32B. Con-G-2003 140CF.pep (626 a.a.) (SEQ ID NO: 45). Aminoacids in bold identify the junction of the deleted fusion cleavage site.FIG. 32C. CODON-OPTIMIZED Con-G-2003.seq (SEQ ID NO: 46).

FIGS. 33A-33C. FIG. 33A. CONSENSUS_01_AE-2003 (854 a.a.) (SEQ ID NO:47). Amino acid sequence underlined is the fusion domain that is deletedin 140CF design and the “W” underlined is the last amino acid at theC-terminus, all amino acids after the “W” are deleted in the 140CFdesign. FIG. 33B. Con-AE01-2003 140CF.pep (638 a.a.) (SEQ ID NO: 48).Amino acids in bold identify the junction of the deleted fusion cleavagesite. FIG. 33C, CODON-OPTIMIZED Con-AE01-2003.seq. (1945 nt.) (SEQ IDNO: 49).

FIGS. 34A-34C. FIG. 34A. Wild-type subtype A Env. 00KE MSA4076-A(Subtype A, 891 a.a.) (SEQ ID NO: 50). Amino acid sequence underlined isthe fusion domain that is deleted in 140CF design and the “W” underlinedis the last amino acid at the C-terminus, all amino acids after the “W”are deleted in the 140CF design. FIG. 34B. 00KE MSA4076-A 140CF.pep (647a.a.) (SEQ ID NO: 51). Amino acids in bold identify the junction of thedeleted fusion cleavage site. FIG. 34C. CODON-OPTIMIZED 00KE MSA4076-A140CF.seq. (1972 nt.) (SEQ ID NO: 52).

FIGS. 35A-35C. FIG. 35A. Wild-type subtype B. QH0515.1g gp160 (861 a.a.)(SEQ ID NO: 53). Amino acid sequence underlined is the fusion domainthat is deleted in 140CF design and the “W” underlined is the last aminoacid at the C-terminus, all amino acids after the “W” are deleted in the140CF design. FIG. 35B. QH0515.1g 140CF (651 a.a.) (SEQ ID NO: 54).Amino acids in bold identify the junction of the deleted fusion cleavagesite. FIG. 35C. CODON-OPTIMIZED QH0515.1g 140CF.seq (1984 nt.) (SEQ IDNO: 55).

FIGS. 36A-36C. FIG. 36A. Wild-type subtype C. DU123.6 gp160 (854 a.a.)(SEQ ID NO: 56). Amino acid sequence underlined is the fusion domainthat is deleted in 140CF design and the “W” underlined is the last aminoacid at the C-terminus, all amino acids after the “W” are deleted in the140CF design. FIG. 36B. DU123.6 140CF (638 a.a.) (SEQ ID NO: 57). Aminoacids in bold identify the junction of the deleted fusion cleavage site.FIG. 36C. CODON-OPTIMIZED DU123.6 140CF.seq (1945 nt.) (SEQ ID NO: 58).

FIGS. 37A-37C. FIG. 37A. Wild-type subtype CRF01_AE. 97CNGX2F-AE (854a.a.) (SEQ ID NO: 59). Amino acid sequence underlined is the fusiondomain that is deleted in 140CF design and the “W” underlined is thelast amino acid at the C-terminus, all amino acids after the “W” aredeleted in the 140CF design. FIG. 37B. 97CNGX2F-AE 140CF.pep (629 a.a.)(SEQ ID NO: 60). Amino acids in bold identify the junction of thedeleted fusion cleavage site. FIG. 37C. CODON-OPTIMIZED 97CNGX2F-AE140CF.seq (1921 nt.) (SEQ ID NO: 61).

FIGS. 38A-38C. FIG. 38A. Wild-type DRCBL-G (854 a.a.) (SEQ ID NO: 62).Amino acid sequence underlined is the fusion domain that is deleted in140CF design and the “W” underlined is the last amino acid at theC-terminus, all amino acids after the “W” are deleted in the 140CFdesign. FIG. 38B. DRCBL-G 140CF.pep (630 a.a.) (SEQ ID NO: 63). Aminoacids in bold identify the junction of the deleted fusion cleavage site.FIG. 38C. CODON-OPTIMIZED DRCBL-G 140CF.seq (1921 nt.) (SEQ ID NO: 64).

FIGS. 39A and 39B. FIG. 39A. 2003 Con-S Env (SEQ ID NO: 65). FIG. 39B.2003 Con-S Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 72)

FIGS. 40A and 40B. FIG. 40A. 2003 M. Group.Anc Env (SEQ ID NO: 66). FIG.40B. 2003 M. Group.anc Env.seq.opt. (Seq.opt.=codon optimized encodingsequence.) (SEQ ID NO: 67)

FIGS. 41A and 41B. FIG. 41A. 2003 CON_A1 Env (SEQ ID NO: 68). FIG. 41B.2003 CON_A1 Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 70)

FIGS. 42A and 42B. FIG. 42A. 2003 A1.Anc Env (SEQ ID NO: 69). FIG. 42B.2003 A1.anc Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 71)

FIGS. 43A and 43B. FIG. 43A. 2003 CON_A2 Env (SEQ ID NO: 73). FIG. 43B.2003 CON_A2 Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 75)

FIGS. 44A and 44B. FIG. 44A. 2003 CON_B Env (SEQ ID NO: 74). FIG. 44B.2003 CON_B Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 76)

FIGS. 45A and 45B. FIG. 45A. 2003 B.anc Env (SEQ ID NO: 77). FIG. 45B.2003 B.anc Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 79)

FIGS. 46A and 46B. FIG. 46A. 2003 CON_C Env (SEQ ID NO: 78). FIG. 46B.2003 CON_C Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 80)

FIGS. 47A and 47B. FIG. 47A. 2003 C.anc Env (SEQ ID NO: 81). FIG. 47B.2003 C.anc Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 83)

FIGS. 48A and 48B. FIG. 48A. 2003 CON_D Env (SEQ ID NO: 82). FIG. 48B.2003 CON D Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 84)

FIGS. 49A and 49B. FIG. 49A. 2003 CON_F1 Env (SEQ ID NO: 85). FIG. 49B.2003 CON_F1 Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 87)

FIGS. 50A and 50B. FIG. 50A. 2003 CON_F2 Env (SEQ ID NO: 86). FIG. 50B.2003 CON_F2 Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 88)

FIGS. 51A and 51B. FIG. 51A. 2003 CON_G Env (SEQ ID NO: 89). FIG. 51B.2003 CON_G Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 91)

FIGS. 52A and 52B. FIG. 52A. 2003 CON_H Env (SEQ ID NO: 90). FIG. 52B.2003 CON_H Env.seq.opt. (Seq.opt.=codon optimized encoding sequence.)(SEQ ID NO: 92)

FIGS. 53A and 53B. FIG. 53A. 2003_CON_01_AE Env (SEQ ID NO: 93). FIG.53B. 2003_CON_01_AE Env.seq.opt. (Seq.opt.=codon optimized encodingsequence.) (SEQ ID NO: 95)

FIGS. 54A and 54B. FIG. 54A. 2003 CON_02_AG Env (SEQ ID NO: 94). FIG.54B. 2003 CON_02_AG Env.seq.opt. (Seq.opt.=codon optimized encodingsequence.) (SEQ ID NO: 96)

FIGS. 55A and 55B. FIG. 55A. 2003 CON_03_AB Env (SEQ ID NO: 97). FIG.55B. 2003 CON_03_AB Env.seq.opt. (Seq.opt.=codon optimized encodingsequence.) (SEQ ID NO: 99)

FIGS. 56A and 56B. FIG. 56A. 2003 CON_04_CPX Env (SEQ ID NO: 98). FIG.56B. 2003 CON_04_CPX Env.seq.opt. (Seq.opt.=codon optimized encodingsequence.) (SEQ ID NO: 100)

FIGS. 57A and 57B. FIG. 57A. 2003 CON_06_CPX Env (SEQ ID NO: 101). FIG.57B. 2003 CON_06_CPX Env.seq.opt. (Seq.opt.=codon optimized encodingsequence.) (SEQ ID NO: 103)

FIGS. 58A and 58B. FIG. 58A. 2003 CON_08_BC Env (SEQ ID NO: 102). FIG.58B. 2003 CON_08_BC Env.seq.opt. (Seq.opt.=codon optimized encodingsequence.) (SEQ ID NO: 104)

FIGS. 59A and 59B. FIG. 59A. 2003 CON_10_CD Env (SEQ ID NO: 105). FIG.59B. 2003 CON_10_CD Env.seq.opt. (Seq.opt.=codon optimized encodingsequence.) (SEQ ID NO: 107)

FIGS. 60A and 60B. FIG. 60A. 2003 CON_11_CPX Env (SEQ ID NO: 106). FIG.60B. 2003 CON_11_CPX Env.seq.opt. (Seq.opt.=codon optimized encodingsequence.) (SEQ ID NO: 108)

FIGS. 61A and 61B. FIG. 61A. 2003 CON_12_BF Env (SEQ ID NO: 109). FIG.61B. 2003 CON_12_BF Env.seq.opt. (Seq.opt.=codon optimized encodingsequence.) (SEQ ID NO: 111)

FIGS. 62A and 62B. FIG. 62A. 2003 CON_14_BG Env (SEQ ID NO: 110). FIG.62B, 2003 CON_14_BG Env.seq.opt. (Seq.opt.=codon optimized encodingsequence.) (SEQ ID NO: 112)

FIGS. 63A and 63B. FIG. 63A. 2003_CON_S gag.PEP (SEQ ID NO: 113). FIG.63B. 2003_CON_S gag.OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 114)

FIGS. 64A and 64B. FIG. 64A. 2003_M.GROUP.anc gag.PEP (SEQ ID NO: 115).FIG. 64B. 2003_M.GROUP.anc gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 116)

FIGS. 65A-65D. FIG. 65A. 2003_CON_A1 gag.PEP (SEQ ID NO: 117). FIG. 65B.2003_CON_A1 gag.OPT (SEQ ID NO: 118). FIG. 65C. 2003_A1.anc gag.PEP (SEQID NO: 119). FIG. 65D. 2003_A1.anc gag.OPT (SEQ ID NO: 120). (OPT=codonoptimized encoding sequence.)

FIGS. 66A and 66B. FIG. 66A. 2003_CON_A2 gag.PEP (SEQ ID NO: 121), FIG.66B. 2003_CON_A2 gag.OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 122)

FIGS. 67A-67D. FIG. 67A. 2003_CON_B gag.PEP (SEQ ID NO: 123). FIG. 67B.2003_CON_B gag.OPT (SEQ ID NO: 124). FIG. 67C. 2003_B, anc gag.PEP (SEQID NO: 125). FIG. 67D. 2003_B.anc gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 126)

FIGS. 68A-68D. FIG. 68A. 2003_CON_C gag.PEP (SEQ ID NO: 127). FIG. 68B.2003_CON_C gag.OPT (SEQ ID NO: 128). FIG. 68C. 2003_C.anc.gag.PEP (SEQID NO: 129). FIG. 68D. 2003_C.anc.gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 130)

FIGS. 69A and 69B. FIG. 69A. 2003_CON_D gag.PEP (SEQ ID NO: 131). FIG.69B. 2003_CON_D gag.OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 132)

FIGS. 70A and 70B. FIG. 70A. 2003_CON_F gag.PEP (SEQ ID NO: 133). FIG.70B. 2003_CON_F gag.OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 134)

FIGS. 71A and 71B. FIG. 71A. 2003_CON_G gag.PEP (SEQ ID NO: 135). FIG.71B. 2003_CON_G gag.OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 136)

FIGS. 72A and 72B. FIG. 72A. 2003_CON_H gag.PEP (SEQ ID NO: 137). FIG.72B. 2003_CON_H gag.OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 138)

FIGS. 73A and 73B. FIG. 73A. 2003_CON_K gag.PEP (SEQ ID NO: 139). FIG.73B. 2003_CON_K gag.OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 140)

FIGS. 74A and 74B. FIG. 74A. 2003_CON_01_AE gag.PEP (SEQ ID NO: 141).FIG. 7B. 2003_CON_01_AE gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 142)

FIGS. 75A and 75B. FIG. 75A. 2003_CON_02_AG gag.PEP (SEQ ID NO: 143).FIG. 75B. 2003_CON_02_AG gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 144)

FIGS. 76A and 76B. FIG. 76A. 2003_CON_03_ABG gag.PEP (SEQ ID NO: 145).FIG. 76B. 2003_CON_03_ABG gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 146)

FIGS. 77A and 77B. FIG. 77A. 2003_CON_04_CFX gag.PEP (SEQ ID NO: 147).FIG. 77B. 2003_CON_04_CFX gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 148)

FIGS. 78A and 78B. FIG. 78A. 2003_CON_06_CPX gag.PEP (SEQ ID NO: 150).FIG. 78B. 2003_CON_06_CPX gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 151)

FIGS. 79A and 79B. FIG. 79A. 2003_CON_07_BC gag.PEP (SEQ ID NO: 152).FIG. 79B. 2003_CON_07_BC gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 153)

FIGS. 80A and 80B. FIG. 80A. 2003_CON_08_BC gag.PEP (SEQ ID NO: 154).FIG. 80B. 2003_CON_08_BC gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 155)

FIGS. 81A and 81B. FIG. 81A. 2003_CON_10_CD gag.PEP (SEQ ID NO: 156).FIG. 81B. 2003_CON_10_CD gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 157)

FIGS. 82A and 82B. FIG. 82A. 2003_CON_11_CPX gag.PEP (SEQ ID NO: 158).FIG. 82B. 2003_CON_11_CPX gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 159)

FIGS. 83A and 83B. FIG. 83A. 2003_CON_12_BF.gag.PEP (SEQ ID NO: 160)FIG. 83B. 2003_CON_12_BF.gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 161)

FIGS. 84A and 84B. FIG. 84A. 2003_CON_14_BG gag.PEP (SEQ ID NO: 162).FIG. 84B. 2003_CON_14_BG gag.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 163)

FIGS. 85A and 85B. FIG. 85A. 2003_CONS nef.PEP (SEQ ID NO: 164). FIG.85B. 2003_CONS nef.OPT. (OPT=codon optimized encoding sequence.) (SEQ IDNO: 165)

FIGS. 86A and 86B. FIG. 86A. 2003_M GROUP.anc nef.PEP (SEQ ID NO: 166).FIG. 86B. 2003 M GROUP.anc.nef OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 167)

FIGS. 87A and 87B. FIG. 87A. 2003_CON_A nef.PEP (SEQ ID NO: 168). FIG.87B. 2003_CON_A nef OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 169)

FIGS. 88A-88D. FIG. 88A. 2003_CON_A1 nef.PEP (SEQ ID NO: 170). FIG. 88B.2003_CON_A1 nef.OPT (SEQ ID NO: 171). FIG. 88C. 2003_A1.anc nef.PEP (SEQID NO: 172). FIG. 88D. 2003_A1.anc nef.OPT. (OPT=codon optimizedencoding sequence.) (SEQ ID NO: 173)

FIGS. 89A and 89B. FIG. 89A. 2003_CON_A2 nef.PEP (SEQ ID NO: 174). FIG.89B. 2003_CON_A2 nef.OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 175)

FIGS. 90A-90D. FIG. 90A. 2003_CON_B nef PEP (SEQ ID NO: 176). FIG. 90B.2003_CON-B nef OPT (SEQ ID NO: 177). FIG. 90C. 2003_B.anc nef.PEP (SEQID NO: 178). FIG. 90D. 2003_B.anc nef.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 179)

FIGS. 91A and 91B. FIG. 91A. 2003_CON_02_AG nef.PEP (SEQ ID NO: 180).FIG. 91B. 2003_CON_02_AG nef.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 181)

FIGS. 92A-92D. FIG. 92A. 2003_CON_C nef PEP (SEQ ID NO: 182). FIG. 92B.2003_CON_C nef OPT (SEQ ID NO: 183). FIG. 92C. 2003_C.anc nef PEP (SEQID NO: 184). FIG. 92D. 2003_C.anc nef.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 185)

FIGS. 93A and 93B. FIG. 93A. 2003_CON_D nef.PEP (SEQ ID NO: 186). FIG.93B. 2003_CON_D nef OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 187)

FIGS. 94A and 94B. FIG. 94A. 2003_CON_F1 nef PEP (SEQ ID NO: 188). FIG.94B. 2003_CON_F1 nef OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 189)

FIGS. 95A and 95B. FIG. 95A. 2003_CON_F2 nef.PEP (SEQ ID NO: 190). FIG.95B. 2003_CON_F2 nef.OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 191)

FIGS. 96A and 96B. FIG. 96A. 2003_CON_G nef.PEP (SEQ ID NO: 192). FIG.96B. 2003_CON_G nef OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 193)

FIGS. 97A and 97B. FIG. 97A. 2003_CON_H nef.PEP (SEQ ID NO: 194). FIG.97B. 2003_CON_H nef.OPT. (OPT=codon optimized encoding sequence.) (SEQID NO: 195)

FIGS. 98A and 98B. FIG. 98A. 2003_CON_01_AE nef.PEP (SEQ ID NO: 196).FIG. 98B. 2003_CON_01_AE nef OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 197)

FIGS. 99A and 99B. FIG. 99A. 2003_CON_03_AE nef.PEP (SEQ ID NO: 198).FIG. 99B. 2003_CON_03_AE nef OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 199)

FIGS. 100A and 100B. FIG. 100A. 2003_CON_04_CFX nef.PEP (SEQ ID NO:200). FIG. 100B. 2003_CON_04_CFX nef.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 201)

FIGS. 101A and 101B. FIG. 101A. 2003_CON_06_CFX nef.PEP (SEQ ID NO:202). FIG. 101B. 2003_CON_06_CFX nef.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 203)

FIGS. 102A and 102B. FIG. 102A. 2003_CON_08_BC nef.PEP (SEQ ID NO: 204).FIG. 102B. 2003_CON_08_BC nef OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 205)

FIGS. 103A and 103B. FIG. 103A. 2003_CON_10_CD nef.PEP (SEQ ID NO: 206).FIG. 103B. 2003_CON_10_CD nef.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 207)

FIGS. 104A and 104B. FIG. 104A. 2003_CON_11_CFX nef.PEP (SEQ ID NO:208). FIG. 104B. 2003_CON_11_CFX nef.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 209)

FIGS. 105A and 105B. FIG. 105A. 2003_CON_12_BF nef.PEP (SEQ ID NO: 210).FIG. 105B. 2003_CON_12_BF nef.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 211)

FIGS. 106A and 106B. FIG. 106A. 2003_CON_14_BG nef.PEP (SEQ ID NO: 212).FIG. 106B. 2003_CON_14_BG nef.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 213)

FIGS. 107A and 107B. FIG. 107A. 2003_CON_S pol.PEP (SEQ ID NO: 214).FIG. 107B. 2003_CON_S pol.OPT. (OPT=codon optimized encoding sequence.)(SEQ ID NO: 215)

FIGS. 108A and 108B. FIG. 108A. 2003_M GROUP anc pol.PEP (SEQ ID NO:216). FIG. 108B. 2003_M.GROUP anc pol.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 218)

FIGS. 109A-109D. FIG. 109A. 2003_CON_A1 pol.PEP (SEQ ID NO: 217). FIG.109B. 2003_CON_A1 pol.OPT (SEQ ID NO: 219). FIG. 109C. 2003_A1.ancpol.PEP (SEQ ID NO: 220). FIG. 109D. 2003_A1.anc pol.OPT (SEQ ID NO:221). (OPT=codon optimized encoding sequence.)

FIGS. 110A and 110B. FIG. 110A. 2003_CON_A2 pol.PEP (SEQ ID NO: 222).FIG. 110B. 2003_CON_A2 pol.OPT. (OPT=codon optimized encoding sequence.)(SEQ ID NO: 224)

FIGS. 111A-111D. FIG. 111A. 2003_CON_B pol.PEP (SEQ ID NO: 223). FIG.111B. 2003_CON_B pol.OPT (SEQ ID NO: 225). FIG. 111C. 2003_B.anc pol.PEP(SEQ ID NO: 226). FIG. 111D. 2003_B.anc pol.OPT (SEQ ID NO: 227).(OPT=codon optimized encoding sequence.)

FIGS. 112A-112D. FIG. 112A. 2003_CON_C pol.PEP (SEQ ID NO: 228). FIG.112B. 2003_CON_C pol.OPT (SEQ ID NO: 229). FIG. 112C. 2003_C.anc pol.PEP(SEQ ID NO: 230). FIG. 112D. 2003_C.anc pol.OPT. (OPT=codon optimizedencoding sequence.) (SEQ ID NO: 231)

FIGS. 113A and 113B. FIG. 113A. 2003_CON_D pol.PEP (SEQ ID NO: 232).FIG. 113B. 2003_CON_D pol.OPT. (OPT=codon optimized encoding sequence.)(SEQ ID NO: 224)

FIGS. 114A and 114B. FIG. 114A. 2003_CON_F1 pol.PEP (SEQ ID NO: 233).FIG. 114B. 2003_CON_F1 pol.OPT. (OPT=codon optimized encoding sequence.)(SEQ ID NO: 235)

FIGS. 115A and 115B. FIG. 115A. 2003_CON_F2 pol.PEP (SEQ ID NO: 236).FIG. 115B. 2003_CON_F2 pol.OPT. (OPT=codon optimized encoding sequence.)(SEQ ID NO: 238)

FIGS. 116A and 116B. FIG. 116A. 2003_CON_G pol.PEP (SEQ ID NO: 237).FIG. 116B. 2003_CON_G pol.OPT. (OPT=codon optimized encoding sequence.)(SEQ ID NO: 239)

FIGS. 117A and 117B. FIG. 117A. 2003_CON_H pol.PEP (SEQ ID NO: 240).FIG. 117B. 2003_CON_H pol.OPT. (OPT=codon optimized encoding sequence.)(SEQ ID NO: 242)

FIGS. 118A and 118B. FIG. 118A. 2003_CON_01_AE pol.PEP (SEQ ID NO: 241).FIG. 118B. 2003_CON_01_AE pol.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 243)

FIGS. 119A and 119B. FIG. 119A. 2003_CON_02_AG pol.PEP (SEQ ID NO: 244).FIG. 119B. 2003_CON_02_AG pol.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 246)

FIGS. 120A and 120B. FIG. 120A. 2003_CON_03_AB pol.PEP (SEQ ID NO: 245).FIG. 120B. 2003_CON_03_AB pol.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 247)

FIGS. 121A and 121B. FIG. 121A. 2003_CON_04_CPX pol.PEP (SEQ ID NO:248). FIG. 121B. 2003_CON_04_CPX pol.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 250)

FIGS. 122A and 122B. FIG. 122A. 2003_CON_06_CPX pol.PEP (SEQ ID NO:249). FIG. 122B. 2003_CON06_CPX pol.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 251)

FIGS. 123A and 123B. FIG. 123A. 2003_CON_08_BC pol.PEP (SEQ ID NO: 252).FIG. 123B. 2003_CON_08_BC pol.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 254)

FIGS. 124A and 124B. FIG. 124A. 2003_CON_10_CD pol.PEP (SEQ ID NO: 253).FIG. 124B. 2003_CON_10_CD pol.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 255)

FIGS. 125A and 125B. FIG. 125A. 2003_CON_11_CPX pol.PEP (SEQ ID NO:256). FIG. 125B. 2003_CON_11_CPX pol.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 258)

FIGS. 126A and 126B. FIG. 126A. 2003_CON_12_BF pol.PEP (SEQ ID NO: 257).FIG. 126B. 2003_CON_12_BF pol.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 259)

FIGS. 127A and 127B. FIG. 127A. 2003_CON_14_BG pol.PEP (SEQ ID NO: 260).FIG. 127B. 2003_CON_14_BG pol.OPT. (OPT=codon optimized encodingsequence.) (SEQ ID NO: 261)

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an immunogen that induces antibodiesthat neutralize a wide spectrum of human immunodeficiency virus (HIV)primary isolates and/or that induces a T cell response. The immunogencomprises at least one consensus or ancestral immunogen (e.g., Env, Gag,Nef or Poly, or portion or variant thereof. The invention also relatesto nucleic acid sequences encoding the consensus or ancestral immunogen,or portion or variant thereof. The invention further relates to methodsof using both the immunogen and the encoding sequences. While theinvention is described in detail with reference to specific consensusand ancestral immunogens (for example, to a group M consensus Env), itwill be appreciated that the approach described herein can be used togenerate a variety of consensus or ancestral immunogens (for example,envelopes for other HIV-1 groups (e.g., N and O)).

In accordance with one embodiment of the invention, a consensus env genecan be constructed by generating consensus sequences of env genes foreach subtype of a particular HIV-1 group (group M being classified intosubtypes A-D, F-H, J an K), for example, from sequences in the LosAlamos HIV Sequence Database (using, for example, MASE (Multiple AlignedSequence Editor)). A consensus sequence of all subtype consensuses canthen be generated to avoid heavily sequenced subtypes (Gaschen et al,Science 296:2354-2360 (2002), Korber et al, Science 288:1789-1796(2000)). In the case of the group M consensus env gene described inExample 1 (designated CON6), five highly variable regions from aCRF08_BC recombinant strain (98CN006) (V1, V2, V4, V5 and a region incytoplasmic domain of gp41) are used to fill in the missing regions inthe sequence (see, however, corresponding regions for Con-S). For highlevels of expression, the codons of consensus or ancestral genes can beoptimized based on codon usage for highly expressed human genes. (Haaset al, Curr. Biol. 6:315-324 (2000), Andre et al, J. Virol. 72:1497-1503(1998)).

With the Year 1999 consensus group M env gene, CONE, it has beenpossible to demonstrate induction of superior T cell responses by CONEversus wild-type B and C env by the number of ELISPOT γ-interferonspleen spot forming cells and the number of epitopes recognized in twostrains of mice (Tables 1 and 2 show the data in BALB/c mice). Theability of CON6 Env protein to induce neutralizing antibodies to HIV-1primary isolates has been compared to that of several subtype B Env. Thetarget of neutralizing antibodies induced by CON6 includes several non-BHIV-1 strains.

TABLE 1 T cell epitope mapping of CON6, JRFL and96ZM651 Env immunogen in BALB/c mice.Table 1 discloses SEQ ID NOS: 262-287,respectively, in order of appearance. Immunogen JRFL 96ZM651 T cellPeptide CON6 (B) (C) response CON 6 (group M consensus)   16DTEVHNVWATHACVP + + CD4   48 KNSSEYYRLINCNTS + + CD4   49    EYYRLINCNTSAITQ   53 CPKVSFEPIPIHYCA + CD4   54     SFEPIPIHYCAPAGF  62 NVSTVQCTHGIKPVV + CD4  104 ETITLPCRIKQIINM + CD8  105    LPCRIKQIINMWQGV  130 GIVQQQSNLLRAIEA + CD4  131    VQQSNLLRAIEAQQHL 134 AQQHLLQLTVWGIKQLQ + CD4  135      LQLTVWGIKQLQARVL Subtype B (MN)6223 AKAYDTEVHNVWATQ + CD4 6224     DTEVHNVWATQACVP 6261ACPKISFEPIPIHYC + CD4 6262     ISFEPIPIHYCAPAG 6286 RKRIHIGPGRAFYTT +CD8 6287     HIGPGRAFYTTKNII 6346 IVQQQNNLLRAIEAQ + CD4 6347    QNNLLRAIEAQQHML Subtype C (Chn19) 4834 VPVWKEAKTTLFCASDAKSY + CD44836 GKEVHNVWATHACVPTDPNP + + CD4 4848 SSENSSEYYRLINCNTSAIT + + CD4 4854STVQCTHGIKPVVSTQLLLN + CD4 4884 QQSNLLRAIEAQQHLLQLTV + CD4 4885AQQHLLQLTVWGIKQLQTRV + CD4

TABLE 2 T cell epitope mapping of CON6.gp120 immunogen in C57BL/6 mice.Table 2 discloses SEQ ID NOS: 288-304,respectively, in order of appearance. Peptide Peptide sequenceT cell response CON 6 (consensus)    2 GIQRNCQHLWRWGTM CD8    3    NCQHLWRWGTMILGM   16 DTEVENVWATHACVP CD4   53 CPKVSFEPIPIHYCA CD4  97 FYCNTSGLFNSTWMF CD8   99 FNSTWMFNGTYMFNG CD8 Subtype B (MN) 6210GIRRNYQHWWGWGTM CD8 6211     NYQHWWGWGTMLLGL 6232 NMWKNNMVEQMHEDI CD46262 ISFEPIPIHYCAPAG CD4 6290 NIIGTIRQAHCNISR CD4 6291    TIRQAHCNISRAKWN Subtype C (Chn 19) 4830 MRVTGIRKNYQHLWRWGTML CD85446 RWGTMLLGMLMICSAAEN CD8 4836 GKEVENVWATHACVPTDPNP CD4 4862GDIRQAHCNISKDKWNETLQ CD4 4888 LLGIWGCSGKLICTTTVPWN CD8

For the Year 2000 consensus group M env gene, Con-S, the Con-S envelopehas been shown to be as immunogenic as the CON6 envelope gene in T cellγ interferon ELISPOT assays in two strains of mice (the data for C57BL/6are shown in FIG. 27). Furthermore, in comparing CON6 and Con-S gp140Envs as protein immunogens for antibody in guinea pigs (Table 3), bothgp140 Envs were found to induce antibodies that neutralized subtype Bprimary isolates. However, Con-S gp140 also induced robustneutralization of the subtype C isolates TV-1 and DU 123 as well as onesubtype A HIV-1 primary isolate, while CON6 did not.

TABLE 3 Ability of Group M Consensus CON6 and Con-S Envs to InduceNeutralization of HIV-1 Primary Isolates CON6 gp140CF CON6 gp140 CFICONS gp140 CFI HIV-1 Isolate Guinea Pig Number (Subtype) 770 771 772 775781 783 784 786 776 777 778 780 BX08(B) 520 257 428 189 218 164 >540199 >540 >540 >540 >5 QH0692 (B) 46 55 58 77 <20 91 100 76 109 <20 <20<20 SS1196(B) 398 306 284 222 431 242 >540 351 >540 296 >540 >540JRLFL(B) <20 <20 <20 <20 <20 169 <20 <20 <20 <20 <20 <20 BG1168(B) <20<20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 3988(B) <20 <20 <20 <20 <20<20 <20 <20 <20 <20 <20 <20 6101(B) <20 <20 <20 <20 <20 <20 <20 <20 <20<20 <20 <20 TV-1(C) <20 <20 <20 <20 <20 <20 <20 <20 356 439 >540 >540DU123(C) <20 <20 71 74 <20 72 <20 <20 176 329 387 378 DU172(C) <20 <2096 64 <20 <20 <20 <20 <20 235 <20 213 ZM18108.6(C) ND ND ND ND <20 <20<20 <20 84 61 86 43 ZM14654.7(C) ND ND ND ND <20 <20 <20 <20 <20 <20 30<20 DU151(C) <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 DU422(C)<20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 DU156(C) <20 <20 <20 <20<20 <20 <20 <20 <20 <20 <20 <20 92RWO20(A) <20 <20 <20 <20 <20 <20 <20<20 116 204 95 117 92UG037(A) <20 <20 30 <20 <20 44 <20 <20 <20 <20 <20<2 ‡ 50% Neutralization titers after 4th or 5th immunizations Year 2000Con-S 140CFI.ENV sequence is shown in FIG. 26A. Gp140 CFI refers to anHIV-1 envelope design in which the cleavage-site is deleted (c), thefusion-site is deleted (F) and the gp41 immunodominant region is deleted(I), in addition to the deletion of transmembrane and cytoplasmicdomains. The codon-optimized Year 2000 Con-S 140 CFI sequence is shownin FIG. 26B.

As the next iteration of consensus immunogens, and in recognition of thefact that a practical HIV-1 immunogen can be a polyvalent mixture ofeither several subtype consensus genes, a mixture of subtype andconsensus genes, or a mixture of centralized genes and wild type genes,a series of 11 subtype consensus, and wild type genes have been designedfrom subtypes A, B, C, CRF AE01, and G as well as a group M consensusgene from Year 2003 Los Alamos National Database sequences. The wildtype sequences were chosen either because they were known to come fromearly transmitted HIV-1 strains (those strains most likely to benecessary to be protected against by a vaccine) or because they were themost recently submitted strains in the database of that subtype. Thesenucleotide and amino acid sequences are shown in FIGS. 28-38 (for all140CF designs shown, 140CF gene can be flanked with the 5′ sequence“TTCAGTCGACGGCCACC” (SEQ ID NO: 305) that contains a Kozak sequence(GCCACCATGG/A) (SEQ ID NO: 306) and SalI site and 3′ sequence ofTAAAGATCTTACAA (SEQ ID NO: 307) containing stop codon and BglII size).Shown in FIGS. 39-62 are 2003 centralized (consensus and ancestral)HIV-1 envelope proteins and the codon optimized gene sequences.

Major differences between CONE gp140 (which does not neutralizenon-clade B HIV strains) and Con-S gp140 (which does induce antibodiesthat neutralize non-clade B HIV strains) are in Con-S V1, V2, V4 and V5regions. For clade B strains, peptides of the V3 region can induceneutralizing antibodies (Haynes et al, J. Immunol. 151:1646-1653(1993)). Thus, construction of Th-V1, Th-V2, Th-V4, Th-V5 peptides canbe expected to give rise to the desired broadly reactive anti-non-cladeB neutralizing antibodies. Therefore, the Th-V peptides set forth inTable 4 are contemplated for use as a peptide immunogen(s) derived fromCon-S gp140. The gag Th determinant (GTH, Table 4) or any homologous GTHsequence in other HIV strains, can be used to promote immunogenicity andthe C4 region of HIV gp120 can be used as well (KQIINMWQVVGKAMYA) (SEQID NO: 308) or any homologous C4 sequence from other HIV strains (Hayneset al, J. Immunol. 151:1646-1653 (1993)). Con-S V1, V2, V4, V5 peptideswith an N-terminal helper determinant can be used singly or together,when formulated in a suitable adjuvant such as Corixa's RC529 (Baldridgeet al, J. Endotoxin Res. 8:453-458 (2002)), to induce broadly crossreactive neutralizing antibodies to non-clade B isolates.

TABLE 4  1) GTH Con-S V1 YKRWIILGLNKIVRMYTNVNVTNTTNNT 132-150 EEKGEIKN 2) GTH Con-S V2 YKRWIILGLNKIVRMYTEIRDKKQKVYA 157-189LFYRLDVVPIDDNNNNSSNYR  3) GTH Con-S V3 YKRWIILGLNKIVRMYTRPNNNTRKSIR294-315 IGPGQAFYAT  4) GTH Con-S V4 YKRWIILGLNKIVRMYNTSGLFNSTWIG 381-408NGTKNNNNTNDTITLP  5) GTH Con-S V5 YKRWIILGLNKIVRMYRDGGNNNTNETE 447-466IFRPGGGD  6) GTH Con-6 V1 YKRWIILGLNKIVRMYNVRNVSSNGTET 132-150 DNEEIKN 7) GTH Con-6 V2 YKRWIILGLNKIVRMYTELRDKKQKVYA 157-196LFYRLDVVPIDDKNSSEISGKNSSEYYR  8) GTH-Con 6 V3YKRWIILGLNKIVRMYTRPNNNTRKSIH 301-322 IGPGQAFYAT  9) GTH Con-6 V4YKRWIILGLNKIVRMYNTSGLFNSTWMF 388-418 NGTYMFNGTKDNSETITLP 10 GTH Con 6 V5YKRWIILGLNKIVRMYRDGGNNSNKNKT 457-477 ETFRPGGGD

It will be appreciated that the invention includes portions and variantsof the sequences specifically disclosed herein. For example, forms ofcodon optimized consensus encoding sequences can be constructed asgp140CF, gp140 CFI, gp120 or gp160 forms with either gp120/41 cleaved oruncleaved. For example, and as regards the consensus and ancestralenvelope sequences, the invention encompasses envelope sequences devoidof V3. Alternatively, V3 sequences can be selected from preferredsequences, for example, those described in U.S. application Ser. No.10/431,596 and U.S. Provisional Application No. 60/471,327. In addition,an optimal immunogen for breadth of response can include mixtures ofgroup M consensus gag, pol, nef and env encoding sequences, and as wellas consist of mixtures of subtype consensus or ancestral encodingsequences for gag, pol, nef and env HIV genes. For dealing with regionaldifferences in virus strains, an efficacious mixture can includemixtures of consensus/ancestral and wild type encoding sequences.

A consensus or ancestral envelope of the invention can be been“activated” to expose intermediate conformations of neutralizationepitopes that normally are only transiently or less well exposed on thesurface of the HIV virion. The immunogen can be a “frozen” triggeredform of a consensus or ancestral envelope that makes available specificepitopes for presentation to B lymphocytes. The result of this epitopepresentation is the production of antibodies that broadly neutralizeHIV. (Attention is directed to WO 02/024149 and to theactivated/triggered envelopes described therein.)

The concept of a fusion intermediate immunogen is consistent withobservations that the gp41 HR-2 region peptide, DP178, can capture anuncoiled conformation of gp41 (Furata et al, Nature Struct. Biol. 5:276(1998)), and that formalin-fixed HIV-infected cells can generate broadlyneutralizing antibodies (LaCasse et al, Science 283:357 (1997)).Recently a monoclonal antibody against the coiled-coil region bound to aconformational determinant of gp41 in HR1 and HR2 regions of thecoiled-coil gp41 structure, but did not neutralize HIV (Jiang et al, J.Virol. 10213 (1998)). However, this latter study proved that thecoiled-coil region is available for antibody to bind if the correctantibody is generated.

The immunogen of one aspect of the invention comprises a consensus orancestral envelope either in soluble form or anchored, for example, incell vesicles or in liposomes containing translipid bilayer envelope. Tomake a more native envelope, gp140 or gp160 consensus or ancestralsequences can be configured in lipid bilayers for native trimericenvelope formation. Alternatively, triggered gp160 in aldrithio 1-2inactivated HIV-1 virions can be used as an immunogen. The gp160 canalso exist as a recombinant protein either as gp160 or gp140 (gp140 isgp160 with the transmembrane region and possibly other gp41 regionsdeleted). Bound to gp160 or gp140 can be recombinant CCR5 or CXCR4co-receptor proteins (or their extracellular domain peptide or proteinfragments) or antibodies or other ligands that bind to the CXCR4 or CCR5binding site on gp120, and/or soluble CD4, or antibodies or otherligands that mimic the binding actions of CD4. Alternatively, vesiclesor liposomes containing CD4, CCR5 (or CXCR4), or soluble CD4 andpeptides reflective of CCR5 or CXCR4 gp120 binding sites. Alternatively,an optimal CCR5 peptide ligand can be a peptide from the N-terminus ofCCR5 wherein specific tyrosines are sulfated (Bormier et al, Proc. Natl.Acad. Sci. USA 97:5762 (2001)). The triggered immunogen may not need tobe bound to a membrane but may exist and be triggered in solution.Alternatively, soluble CD4 (sCD4) can be replaced by an envelope (gp140or gp160) triggered by CD4 peptide mimetopes (Vitra et al, Proc. Natl.Acad. Sci. USA 96:1301 (1999)). Other HIV co-receptor molecules that“trigger” the gp160 or gp140 to undergo changes associated with astructure of gp160 that induces cell fusion can also be used. Ligationof soluble HIV gp140 primary isolate HIV 89.6 envelope with soluble CD4(sCD4) induced conformational changes in gp41.

In one embodiment, the invention relates to an immunogen that has thecharacteristics of a receptor (CD4)-ligated consensus or ancestralenvelope with CCR5 binding region exposed but unlike CD4-ligatedproteins that have the CD4 binding site blocked, this immunogen has theCD4 binding site exposed (open). Moreover, this immunogen can be devoidof host CD4, which avoids the production of potentially harmful anti-CD4antibodies upon administration to a host.

The immunogen can comprise consensus or ancestral envelope ligated witha ligand that binds to a site on gp120 recognized by an A32 monoclonalantibodies (mab) (Wyatt et al, J. Virol. 69:5723 (1995), Boots et al,AIDS Res. Hum. Retro. 13:1549 (1997), Moore et al, J. Virol. 68:8350(1994), Sullivan et al, J. Virol. 72:4694 (1998), Fouts et al, J. Virol.71:2779 (1997), Ye et al, J. Virol. 74:11955 (2000)). One A32 mab hasbeen shown to mimic CD4 and when bound to gp120, upregulates (exposes)the CCR5 binding site (Wyatt et al, J. Virol. 69:5723 (1995)). Ligationof gp120 with such a ligand also upregulates the CD4 binding site anddoes not block CD4 binding to gp120. Advantageously, such ligands alsoupregulate the HR-2 binding site of gp41 bound to cleaved gp120,uncleaved gp140 and cleaved gp41, thereby further exposing HR-2 bindingsites on these proteins—each of which are potential targets for anti-HIVneutralizing antibodies.

In a specific aspect of this embodiment, the immunogen comprises solubleHIV consensus or ancestral gp120 envelope ligated with either an intactA32 mab, a Fab2 fragment of an A32 mab, or a Fab fragment of an A32 mab,with the result that the CD4 binding site, the CCR5 binding site and theHR-2 binding site on the consensus or ancestral envelope areexposed/upregulated. The immunogen can comprise consensus or ancestralenvelope with an A32 mab (or fragment thereof) bound or can compriseconsensus or ancestral envelope with an A32 mab (or fragment thereof)bound and cross-linked with a cross-linker such as 0.3% formaldehyde ora heterobifunctional cross-linker such as DTSSP (Pierce ChemicalCompany). The immunogen can also comprise uncleaved consensus orancestral gp140 or a mixture of uncleaved gp140, cleaved gp41 andcleaved gp120. An A32 mab (or fragment thereof) bound to consensus orancestral gp140 and/or gp120 or to gp120 non-covalently bound to gp41,results in upregulation (exposure) of HR-2 binding sites in gp41, gp120and uncleaved gp140. Binding of an A32 mab (or fragment thereof) togp120 or gp140 also results in upregulation of the CD4 binding site andthe CCR5 binding site. As with gp120 containing complexes, complexescomprising uncleaved gp140 and an A32 mab (or fragment thereof) can beused as an immunogen uncross-linked or cross-linked with cross-linkersuch as 0.3% formaldehyde or DTSSP. In one embodiment, the inventionrelates to an immunogen comprising soluble uncleaved consensus orancestral gp140 bound and cross linked to a Fab fragment or whole A32mab, optionally bound and cross-linked to an HR-2 binding protein.

The consensus or ancestral envelope protein triggered with a ligand thatbinds to the A32 mab binding site on gp120 can be administered incombination with at least a second immunogen comprising a secondenvelope, triggered by a ligand that binds to a site distinct from theA32 mab binding site, such as the CCR5 binding site recognized by mab17b. The 17b mab (Kwong et al, Nature 393:648 (1998) available from theAIDS Reference Repository, NIAID, NIH) augments sCD4 binding to gp120.This second immunogen (which can also be used alone or in combinationwith triggered immunogens other than that described above) can, forexample, comprise soluble HIV consensus or ancestral envelope ligatedwith either the whole 17b mab, a Fab2 fragment of the 17b mab, or a Fabfragment of the 17b mab. It will be appreciated that other CCR5 ligands,including other antibodies (or fragments thereof), that result in theCD4 binding site being exposed can be used in lieu of the 17b mab. Thisfurther immunogen can comprise gp120 with the 17b mab, or fragmentthereof, (or other CCR5 ligand as indicated above) bound or can comprisegp120 with the 17b mab, or fragment thereof, (or other CCR5 ligand asindicated above) bound and cross-linked with an agent such as 0.3%formaldehyde or a heterobifunctional cross-linker, such as DTSSP (PierceChemical Company). Alternatively, this further immunogen can compriseuncleaved gp140 present alone or in a mixture of cleaved gp41 andcleaved gp120. Mab 17b, or fragment thereof (or other CCR5 ligand asindicated above) bound to gp140 and/or gp120 in such a mixture resultsin exposure is of the CD4 binding region. The 17b mab, or fragmentthereof, (or other CCR5 ligand as indicated above) gp140 complexes canbe present uncross-linked or cross-linked with an agent such as 0.3%formaldehyde or DTSSP.

Soluble HR-2 peptides, such as T649Q26L and DP178, can be added to theabove-described complexes to stabilize epitopes on consensus gp120 andgp41 as well as uncleaved consensus gp140 molecules, and can beadministered either cross-linked or uncross-linked with the complex.

A series of monoclonal antibodies (mabs) have been made that neutralizemany HIV primary isolates, including, in addition to the 17b mabdescribed above, mab IgGlb12 that binds to the CD4 binding site on gp120(Roben et al, J. Virol. 68:482 (1994), Mo et al, J. Virol. 71:6869(1997)), mab 2G12 that binds to a conformational determinant on gp120(Trkola et al, J. Virol. 70:1100 (1996)), and mab 2F5 that binds to amembrane proximal region of gp41 (Muster et al, J. Virol. 68:4031(1994)).

As indicated above, various approaches can be used to “freeze” fusogenicepitopes in accordance with the invention. For example, “freezing” canbe effected by addition of the DP-178 or T-649Q26L peptides thatrepresent portions of the coiled coil region, and that when added toCD4-triggered consensus or ancestral envelope, result in prevention offusion (Rimsky et al, J. Virol. 72:986-993 (1998)). HR-2 peptide boundconsensus or ancestral gp120, gp140, gp41 or gp160 can be used as animmunogen or crosslinked by a reagent such as DTSSP or DSP (Pierce Co.),formaldehyde or other crosslinking agent that has a similar effect.

“Freezing” can also be effected by the addition of 0.1% to 3%formaldehyde or paraformaldehyde, both protein cross-linking agents, tothe complex, to stabilize the CD4, CCR5 or CXCR4, HR-2 peptide gp160complex, or to stabilize the “triggered” gp41 molecule, or both (LaCasseet al, Science 283:357-362 (1999)).

Further, “freezing” of consensus or ancestral gp41 or gp120 fusionintermediates can be effected by addition of heterobifunctional agentssuch as DSP (dithiobis[succimidylproprionate]) (Pierce Co. Rockford,Ill., No. 22585ZZ) or the water soluble DTSSP (Pierce Co.) that use twoNHS esters that are reactive with amino groups to cross link andstabilize the CD4, CCR5 or CXCR4, HR-2 peptide gp160 complex, or tostabilize the “triggered” gp41 molecule, or both.

Analysis of T cell immune responses in immunized or vaccinated animalsand humans shows that the envelope protein is normally not a main targetfor T cell immune response although it is the only gene that inducesneutralizing antibodies. HIV-1 Gag, Pol and Nef proteins induce a potentT cell immune response. Accordingly, the invention includes a repertoireof consensus or ancestral immunogens that can induce both humoral andcellular immune responses. Subunits of consensus or ancestral sequencescan be used as T or B cell immunogens. (See Examples 6 and 7, andFigures referenced therein, and FIGS. 63-127.

The immunogen of the invention can be formulated with a pharmaceuticallyacceptable carrier and/or adjuvant (such as alum) using techniques wellknown in the art. Suitable routes of administration of the presentimmunogen include systemic (e.g. intramuscular or subcutaneous).Alternative routes can be used when an immune response is sought in amucosal immune system (e.g., intranasal).

The immunogens of the invention can be chemically synthesized andpurified using methods which are well known to the ordinarily skilledartisan. The immunogens can also be synthesized by well-knownrecombinant DNA techniques. Nucleic acids encoding the immunogens of theinvention can be used as components of, for example, a DNA vaccinewherein the encoding sequence is administered as naked DNA or, forexample, a minigene encoding the immunogen can be present in a viralvector. The encoding sequence can be present, for example, in areplicating or non-replicating adenoviral vector, an adeno-associatedvirus vector, an attenuated Mycobacterium tuberculosis vector, aBacillus Calmette Guerin (BCG) vector, a vaccinia or Modified VacciniaAnkara (MVA) vector, another pox virus vector, recombinant polio andother enteric virus vector, Salmonella species bacterial vector,Shigella species bacterial vector, Venezuelean Equine Encephalitis Virus(VEE) vector, a Semliki Forest Virus vector, or a Tobacco Mosaic Virusvector. The encoding sequence, can also be expressed as a DNA plasmidwith, for example, an active promoter such as a CMV promoter. Other livevectors can also be used to express the sequences of the invention.Expression of the immunogen of the invention can be induced in apatient's own cells, by introduction into those cells of nucleic acidsthat encode the immunogen, preferably using codons and promoters thatoptimize expression in human cells. Examples of methods of making andusing DNA vaccines are disclosed in U.S. Pat. Nos. 5,580,859, 5,589,466,and 5,703,055.

The composition of the invention comprises an immunologically effectiveamount of the immunogen of this invention, or nucleic acid sequenceencoding same, in a pharmaceutically acceptable delivery system. Thecompositions can be used for prevention and/or treatment ofimmunodeficiency virus infection. The compositions of the invention canbe formulated using adjuvants, emulsifiers, pharmaceutically-acceptablecarriers or other ingredients routinely provided in vaccinecompositions. Optimum formulations can be readily designed by one ofordinary skill in the art and can include formulations for immediaterelease and/or for sustained release, and for induction of systemicimmunity and/or induction of localized mucosal immunity (e.g, theformulation can be designed for intranasal administration). The presentcompositions can be administered by any convenient route includingsubcutaneous, intranasal, oral, intramuscular, or other parenteral orenteral route. The immunogens can be administered as a single dose ormultiple doses. Optimum immunization schedules can be readily determinedby the ordinarily skilled artisan and can vary with the patient, thecomposition and the effect sought.

The invention contemplates the direct use of both the immunogen of theinvention and/or nucleic acids encoding same and/or the immunogenexpressed as minigenes in the vectors indicated above. For example, aminigene encoding the immunogen can be used as a prime and/or boost.

The invention includes any and all amino acid sequences disclosed hereinand, where applicable, CF and CFI forms thereof, as well as nucleic acidsequences encoding same (and nucleic acids complementary to suchencoding sequences).

Certain aspects of the invention can be described in greater detail inthe non-limiting Examples that follows.

Example 1

Artificial HIV-1 Group M Consensus Envelope

Experimental Details

Expression of CONE gp120 and gp140 proteins in recombinant vacciniaviruses (VV). To express and purify the secreted form of HIV-1 CON6envelope proteins, CON6 gp120 and gp140CF plasmids were constructed byintroducing stop codons after the gp120 cleavage site (REKR) (SEQ ID NO:319) and before the transmembrane domain (YIKIFIMIVGGLIGLRIVFAVLSIVN)(SEQ ID NO: 320), respectively. The gp120/gp41 cleavage site and fusiondomain of gp41 were deleted in the gp140CF protein. Both CON6 gp120 andgp140CF DNA constructs were cloned into the pSC65 vector (from BernardMoss, NIH, Bethesda, Md.) at SalI and KpnI restriction enzyme sites.This vector contains the lacZ gene that is controlled by the p7.5promoter. A back-to-back P E/L promoter was used to express CON6 envgenes. BSC-1 cells were seeded at 2×10⁵ in each well in a 6-well plate,infected with wild-type vaccinia virus (WR) at a MOI of 0.1 pfu/cell,and 2 hr after infection, pSC65-derived plasmids containing CON6 envgenes were transfected into the VV-infected cells and recombinant (r) VVselected as described (Moss and Earl, Current Protocols in MolecularBiology, eds, Ausubel et al (John Wiley & Sons, Inc. Indianapolis, Ind.)pp. 16.15.1-16.19.9 (1998)). Recombinant VV that contained the CON6 envgenes were confirmed by PCR and sequencing analysis. Expression of theCON6 envelope proteins was confirmed by SDS-PAGE and Western blot assay.Recombinant CON6 gp120 and gp140CF were purified with agarose galanthusNivalis lectin beads (Vector Labs, Burlingame, Calif.), and stored at70° C. until use. Recombinant VV expressing JRFL (vCB-28) or 96ZM651(vT241R) gp160 were obtained from the NIH AIDS Research and ReferenceReagent Program (Bethesda, Md.).

Monoclonal Antibodies and gp120 Wild-type Envelopes. Human mabs againsta conformational determinant on gp120 (A32), the gp120 V3 loop (F39F)and the CCR5 binding site (17b) were the gifts of James Robinson (TulaneMedical School, New Orleans, La.) (Wyatt et al, Nature 393; 705-711(1998), Wyatt et al, J. Virol. 69:5723-5733 (1995)). Mabs 2F5, 447, b12,2G12 and soluable CD4 were obtained from the NIH AIDS Research andReference Reagent Program (Bethesda, Md.) (Gorny et al, J. Immunol.159:5114-5122 (1997), Nyambi et al, J. Virol. 70:6235-6243 (1996),Purtscher et al, AIDS Res. Hum. Retroviruses 10:1651-1658 (1994), Trkolaet al, J. Virol 70:1100-1108 (1996)). T8 is a murine mab that maps tothe gp120 C1 region (a gift from P. Earl, NIH, Bethesda, Md.). BaL(subtype B), 96ZM651 (subtype C), and 93TH975 (subtype E) gp120s wereprovided by QBI, Inc. and the Division of AIDS, NIH. CHO cell lines thatexpress 92U037 (subtype A) and 93BR029 (subtype F) gp140 (secreted anduncleaved) were obtained from NICBS, England.

Surface Plasmon Resonance Biosensor (SPR) Measurements and ELISA. SPRbiosensor measurements were determined on a BIAcore 3000 instrument(BIAcore Inc., Uppsala, Sweden) instrument and data analysis wasperformed using BIAevaluation 3.0 software (BIAcore Inc, Upsaala,Sweden). Anti-gp120 mabs (T8, A32, 17b, 2G12) or sCD4 in 10 mMNa-acetate buffer, pH 4.5 were directly immobilized to a CM5 sensor chipusing a standard amine coupling protocol for protein immobilization.FPLC purified CON6 gp120 monomer or gp140CF oligomer recombinantproteins were flowed over CM5 sensor chips at concentrations of 100 and300 μg/ml, respectively. A blank in-line reference surface (activatedand de-activated for amine coupling) or non-bonding mab controls wereused to subtract non-specific or bulk responses. Soluble 89.6 gp120 andirrelevant IgG was used as a positive and negative control respectivelyand to ensure activity of each mab surface prior to injecting the CON6Env proteins. Binding of CON6 envelope proteins was monitored inreal-time at 25° C. with a continuous flow of PBS (150 mM NaCl, 0.005%surfactant P20), pH 7.4 at 10-30 μl/min. Bound proteins were removed andthe sensor surfaces were regenerated following each cycle of binding bysingle or duplicate 5-10 μpulses of regeneration solution (10 mMglycine-HCl, pH 2.9). ELISA was performed to determine the reactivity ofvarious mabs to CON6 gp120 and gp140CF proteins as described (Haynes etal, AIDS Res. Hum. Retroviruses 11:211-221 (1995)). For assay of humanmab binding to rgp120 or gp140 proteins, end-point titers were definedas the highest titer of mab (beginning at 20 μg/ml) at which the mabbound CON6 gp120 and gp140CF Env proteins ≥3 fold over backgroundcontrol (non-binding human mab).

Infectivity and coreceptor usage assays. HIV-1/SG3Δenv and CON6 orcontrol env plasmids were cotransfected into human 293T cells.Pseudotyped viruses were harvested, filtered and p24 concentration wasquantitated (DuPont/NEN Life Sciences, Boston, Mass.). Equal amounts ofp24 (5 ng) for each pseudovirion were used to infect JC53-BL cells todetermine the infectivity (Derdeyn e al, J. Virol. 74:8358-8367 (2000),Wei et al, Antimicrob Agents Chemother. 46:1896-1905 (2002)). JC53-BLcells express CD4, CCR5 and CXCR4 receptors and contain aβ-galactosidase (β-gal) gene stably integrated under the transcriptionalcontrol of an HIV-1 long terminal repeat (LTR). These cells can be usedto quantify the infectious titers of pseudovirion stocks by staining forβ-gal expression and counting the number of blue cells (infectiousunits) per microgram of p24 of pseudovirons (IU/μg p24) (Derdeyn e al,J. Virol. 74:8358-8367 (2000), Wei et al, Antimicrob Agents Chemother.46:1896-1905 (2002)). To determine the coreceptor usage of the CON6 envgene, JC53BL cells were treated with 1.2 μM AMD3100 and 4 μM TAK-799 for1 hr at 37° C. then infected with equal amounts of p24 (5 ng) of eachEnv pseudotyped virus. The blockage efficiency was expressed as thepercentage of the infectious units from blockage experiments compared tothat from control culture without blocking agents. The infectivity fromcontrol group (no blocking agent) was arbitrarily set as 100%.

Immunizations. All animals were housed in the Duke University AnimalFacility under AALAC guidelines with animal use protocols approved bythe Duke University Animal Use and Care Committee. Recombinant CON6gp120 and gp140CF glycoproteins were formulated in a stable emulsionwith RIBI-CWS adjuvant based on the protocol provided by themanufacturer (Sigma Chemical Co., St. Louis, Mo.). For induction ofanti-envelope antibodies, each of four out-bred guinea pigs (HarlanSprague, Inc., Chicago, Ill.) was given 100 μg either purified CON6gp120 or gp140CF subcutaneously every 3 weeks (total of 5immunizations). Serum samples were heat-inactivated (56° C., 1 hr), andstored at −20° C. until use.

For induction of anti-envelope T cell responses, 6-8 wk old femaleBALB/c mice (Frederick Cancer Research and Developmental Center, NCI,Frederick, Md.) were immunized i.m. in the quadriceps with 50 μg plasmidDNA three times at a 3-week interval. Three weeks after the last DNAimmunization, mice were boosted with 10⁷ PFU of rVV expressing Envproteins. Two weeks after the boost, all mice were euthanized andspleens were removed for isolation of splenocytes.

Neutralization assays. Neutralization assays were performed using eithera MT-2 assay as described in Bures et al, AIDS Res. Hum. Retroviruses16:2019-2035 (2000), a luciferase-based multiple replication cycle HIV-1infectivity assay in 5.25.GFP.Luc.M7 cells using a panel of HIV-1primary isolates (Bures et al, AIDS Res. Hum. Retroviruses 16:2019-2035(2000), Bures et al, J. Virol. 76:2233-2244 (2002)), or a syncytium(fusion from without) inhibition assay using inactivated HIV-1 virions(Rossio et al, J. Virol. 72:7992-8001 (1998)). In the luciferase-basedassay, neutralizing antibodies were measured as a function of areduction in luciferase acitivity in 5.25.EGFP.Luc.M7 cells provided byNathaniel R. Landau, Salk Institute, La Jolla, Calif. (Brandt et al, J.Biol. Chem. 277:17291-17299 (2002)). Five hundred tissue cultureinfectious dose 50 (TCID₅₀) of cell-free virus was incubated withindicated serum dilutions in 150 μl (1 hr, at 37° C.) in triplicate in96-well flat-bottom culture plates. The 5.25.EGFP.Luc.M7 cells weresuspended at a density of 5×10⁵/ml in media containing DEAE dextran (10μg/ml). Cells (100 μl) were added and until 10% of cells in controlwells (no test serum sample) were positive for GFP expression byfluorescence microscopy. At this time the cells were concentrated 2-foldby removing one-half volume of media. A 50 μl suspension of cells wastransferred to 96-well white solid plates (Costar, Cambridge, Mass.) formeasurement of luciferase activity using Bright-Glo™ substrate (Promega,Madison, Wis.) on a Wallac 1420 Multilabel Counter (PerkinElmer LifeSciences, Boston, Mass.). Neutralization titers in the MT-2 andluciferase assays were those where ≥50% virus infection was inhibited.Only values that titered beyond 1:20 (i.e. >1:30) were consideredsignificantly positive. The syncytium inhibition “fusion from without”assay utilized HIV-1 aldrithiol-2 (AT-2) inactivated virions from HIV-1subtype B strains ADA and AD8 (the gift of Larry Arthur and JeffreyLifson, Frederick Research Cancer Facility, Frederick, Md.) added toSupT1 cells, with syncytium inhibition titers determined as those titerswhere ≥90% of syncytia were inhibited compared to prebleed sera.

Enzyme linked immune spot (ELISPOT) assay. Single-cell suspensions ofsplenocytes from individual immunized mice were prepared by mincing andforcing through a 70 μm Nylon cell strainer (BD Labware, Franklin Lakes,N.J.). Overlapping Env peptides of CON6 gp140 (159 peptides, 15mersoverlapping by 11) were purchased from Boston Bioscence, Inc (Royal Oak,Mich.). Overlapping Env peptides of MN gp140 (subtype B; 170 peptides,15mers overlapping by 11) and Chn19 gp140 (subtype C; 69 peptides,20mers overlapping by 10) were obtained from the NIH AIDS Research andReference Reagent Program (Bethesda, Md.). Splenocytes (5 mice/group)from each mouse were stimulated in vitro with overlapping Env peptidespools from CON6, subtype B and subtype C Env proteins. 96-well PVDFplates (MultiScreen-IP, Millipore, Billerica, Mass.) were coated withanti-IFN-γ mab (5 μg/ml, AN18; Mabtech, Stockholm, Sweden). After theplates were blocked at 37° C. for 2 hr using complete Hepes bufferedRPMI medium, 50 μl of the pooled overlapping envelope peptides (13 CON6and MN pools, 13-14 peptides in each pool; 9 Chn19 pool, 7-8 peptide ineach pool) at a final concentration of 5 μg/ml of each were added to theplate. Then 50 μl of splenocytes at a concentration of 1.0×10⁷/ml wereadded to the wells in duplicate and incubated for 16 hr at 37° C. with5% CO₂. The plates were incubated with 100 μl of a 1:1000 dilution ofstreptavidin alkaline phosphatase (Mabtech, Stockholm, Sweden), andpurple spots developed using 100 μl of BCIP/NBT (Plus) AlkalinePhosphatase Substrate (Moss, Pasadena, Md.). Spot forming cells (SFC)were measured using an Immunospot counting system (CTL Analyzers,Cleveland, Ohio). Total responses for each envelope peptide pool areexpressed as SFCs per 10⁶ splenocytes.

Results

CON6 Envelope Gene Design, Construction and Expression. An artificialgroup M consensus env gene (CON6) was constructed by generatingconsensus sequences of env genes for each HIV-1 subtype from sequencesin the Los Alamos HIV Sequence Database, and then generating a consensussequence of all subtype consensuses to avoid heavily sequenced subtypes(Gaschen et al, Science 296:2354-2360 (2002), Korber et al, Science288:1789-1796 (2000)). Five highly variable regions from a CRF08_BCrecombinant strain (98CN006) (V1, V2, V4, V5 and a region in cytoplasmicdomain of gp41) were then used to fill in the missing regions in CON6sequence. The CON6 V3 region is group M consensus (FIG. 1A). For highlevels of expression, the codons of CON6 env gene were optimized basedon codon usage for highly expressed human genes (Haas et al, Curr. Biol.6:315-324 (2000), Andre et al, J. Virol. 72:1497-1503 (1998)). (See FIG.1D.) The codon optimized CON6 env gene was constructed and subclonedinto pcDNA3.1 DNA at EcoR I and BamH I sites (Gao et al, AIDS Res. Hum.Retroviruses, 19:817-823 (2003)). High levels of protein expression wereconfirmed with Western-blot assays after transfection into 293T cells.To obtain recombinant CON6 Env proteins for characterization and use asimmunogens, rVV was generated to express secreted gp120 and uncleavedgp140CF (FIG. 1B). Purity for each protein was ≥90% as determined byCoomassie blue gels under reducing conditions (FIG. 1C).

CD4 Binding Domain and Other Wild-type HIV-1 Epitopes are Preserved onCON6 Proteins. To determine if CON6 proteins can bind to CD4 and expressother wild-type HIV-1 epitopes, the ability of CON6 gp120 and gp140CF tobind soluble(s) CD4, to bind several well-characterized anti-gp120 mabs,and to undergo CD4-induced conformational changes was assayed. First,BIAcore CM5 sensor chips were coated with either sCD4 or mabs to monitortheir binding activity to CON6 Env proteins. It was found that bothmonomeric CON6 gp120 and oligomeric gp140CF efficiently bound sCD4 andanti-gp120 mabs T8, 2G12 and A32, but did not constitutively bind mab17b, that recognizes a CD4 inducible epitope in the CCR5 binding site ofgp120 (FIGS. 2A and 2B). Both sCD4 and A32 can expose the 17b bindingepitope after binding to wild-type gp120 (Wyatt et al, Nature 393;705-711 (1998), Wyatt et al, J. Virol. 69:5723-5733 (1995)). Todetermine if the 17b epitope could be induced on CON6 Envs by eithersCD4 or A32, sCD4, A32 and T8 were coated on sensor chips, then CON6gp120 or gp140CF captured, and mab 17b binding activity monitored. Afterbinding sCD4 or mab A32, both CON6 gp120 and gp140CF were triggered toundergo conformational changes and bound mab 17b (FIGS. 2C and 2D). Incontrast, after binding mab T8, the 17b epitope was not exposed (FIGS.2C and 2D). ELISA was next used to determine the reactivity of a panelof human mabs against the gp120 V3 loop (447, F39F), the CD4 bindingsite (b12), and the gp41 neutralizing determinant (2F5) to CON6 gp120and gp140CF (FIG. 2E). Both CON6 rgp120 and rgp140CF proteins bound wellto neutralizing V3 mabs 447 and F39F and to the potent neutralizing CD4binding site mab b12. Mab 2F5, that neutralizes HIV-1 primary isolatesby binding to a C-terminal gp41 epitope, also bound well to CON6 gp140CF(FIG. 2E).

CON6 env Gene is Biologically Functional and Uses CCR5 as itsCoreceptor. To determine whether CON6 envelope gene is biologicallyfunctional, it was co-transfected with the env-defective SG3 proviralclone into 293T cells. The pseudotyped viruses were harvested and JC53BLcells infected. Blue cells were detected in JC53-BL cells infected withthe CON6 Env pseudovirions, suggesting that CON6 Env protein isbiologically functional (FIG. 3A). However, the infectious titers were1-2 logs lower than that of pseudovirions with either YU2 or NL4-3wild-type HIV-1 envelopes.

The co-receptor usage for the CON6 env gene was next determined. Whentreated with CXCR4 blocking agent AMD3100, the infectivity of NL4-3Env-pseudovirons was blocked while the infectivity of YU2 or CON6Env-pseudovirons was not inhibited (FIG. 3B). In contrast, when treatedwith CCR5 blocking agent TAK-779, the infectivity of NL4-3Env-pseudovirons was not affected, while the infectivity of YU2 or CON6Env-pseudovirons was inhibited. When treated with both blocking agents,the infectivity of all pseudovirions was inhibited. Taken together,these data show that the CON6 envelope uses the CCR5 co-receptor for itsentry into target cells.

Reaction of CON6 gp120 With Different Subtype Sera. To determine ifmultiple subtype linear epitopes are preserved on CON6 gp120, arecombinant Env protein panel (gp120 and gp140) was generated. Equalamounts of each Env protein (100 ng) were loaded on SDS-polyacrylamidegels, transferred to nitrocellulose, and reacted with subtype A throughG patient sera as well as anti-CON6 gp120 guinea pig sera (1:1,000dilution) in Western blot assays. For each HIV-1 subtype, four to sixpatient sera were tested. One serum representative for each subtype isshown in FIG. 4.

It was found that whereas all subtype sera tested showed variablereactivities among Envs in the panel, all group M subtype patient serareacted equally well with CON6 gp120 Env protein, demonstrating thatwild-type HIV-1 Env epitopes recognized by patient sera were wellpreserved on the CON6 Env protein. A test was next made as to whetherCON6 gp120 antiserum raised in guinea pigs could react to differentsubtype Env proteins. It was found that the CON6 serum reacted to itsown and other subtype Env proteins equally well, with the exception ofsubtype A Env protein (FIG. 4).

Induction of T Cell Responses to CON6, Subtype B and Subtype C EnvelopeOverlapping Peptides. To compare T cell immune responses induced by CON6Env immunogens with those induced by subtype specific immunogens, twoadditional groups of mice were immunized with subtype B or subtype CDNAs and with corresponding rVV expressing subtype B or C envelopeproteins. Mice immunized with subtype B (JRFL) or subtype C (96ZM651)Env immunogen had primarily subtype-specific T cell immune responses(FIG. 5). IFN-γ SFCs from mice immunized with JRFL (subtype B) immunogenwere detected after stimulation with subtype B (MN) peptide pools, butnot with either subtype C (Chn19) or CON6 peptide pools. IFN-γ SFCs frommice immunized with 96ZM651 (subtype C) immunogen were detected afterthe stimulation with both subtype C (Chn19) and CON6 peptide pools, butnot with subtype B (MN) peptide pools. In contrast, IFN-γ SFCs wereidentified from mice immunized with CON6 Env immunogens when stimulatedwith either CON6 peptide pools as well as by subtype B or C peptidepools (FIG. 5). The T cell immune responses induced by CON6 gp140appeared more robust than those induced by CON6 gp120. Taken together,these data demonstrated that CON6 gp120 and gp140CF immunogens werecapable of inducing T cell responses that recognized T cell epitopes ofwild-type subtype B and C envelopes.

Induction of Antibodies by Recombinant CON6 gp120 and gp140CF Envelopesthat Neutralize HIV-1 Subtype B and C Primary Isolates. To determine ifthe CON6 envelope immunogens can induce antibodies that neutralize HIV-1primary isolates, guinea pigs were immunized with either CON6 gp120 orgp140CF protein. Sera collected after 4 or 5 immunizations were used forneutralization assays and compared to the corresponding prebleed sera.Two AT-2 inactivated HIV-1 isolates (ADA and AD8) were tested insyncytium inhibition assays (Table 5A). Two subtype B SHIV isolates,eight subtype B primary isolates, four subtype C, and one each subtypeA, D, and E primary isolates were tested in either the MT-2 or theluciferase-based assay (Table 5B). In the syncytium inhibition assay, itwas found that antibodies induced by both CON 6 gp120 and gp140CFproteins strongly inhibited AT-2 inactivated ADA and AD8-inducedsyncytia (Table 5A). In the MT-2 assay, weak neutralization of 1 of 2SHIV isolates (SHIV SF162P3) by two gp120 and one gp140CF sera was found(Table 5B). In the luciferase-based assay, strong neutralization of 4 of8 subtype B primary isolates (BXO8, SF162, SS1196, and BAL) by all gp120and gp140CF sera was found, and weak neutralization of 2 of 8 subtype Bisolates (6101, 0692) by most gp120 and gp140CF sera was found. Noneutralization was detected against HIV-1 PAVO (Table 5B). Next, theCON6 anti-gp120 and gp140CF sera were tested against four subtype CHIV-1 isolates, and weak neutralization of 3 of 4 isolates (DU179,DU368, and S080) was found, primarily by anti-CON6 gp120 sera. Onegp140CF serum, no. 653, strongly neutralized DU179 and weaklyneutralized S080 (Table 5B). Finally, anti-CON6 Env sera stronglyneutralized a subtype D isolate (93ZR001), weakly neutralized a subtypeE (CM244) isolate, and did not neutralize a subtype A (92RW020) isolate.

TABLE 5A Ability of HIV-1 Group M Consensus Envelope CON6 Proteins toInduce Fusion Inhibiting Antibodies Syncytium Inhibition antibody titer¹Guinea Pig No. Immunogen AD8 ADA 646 gp120 270 270 647 gp120 90 90 648gp120 90 270 649 gp120 90 90 Geometric Mean Titer 119 156 650 gp140 270270 651 gp140 90 90 652 gp140 ≥810 810 653 gp140 270 90 Geometric MeanTiter 270 207 ¹Reciprocal serum dilution at which HIV-induced syncytiaof Sup T1 cells was inhibited by >90% compared to pre-immune serum. Allprebleed sera were negative (titer <10).

TABLE 5B Ability of Group M Consensus HIV-1 Envelope CON6 gp120 andgp140CF Proteins to Induce Antibodies that Neutralize HIV PrimaryIsolates CON6 gp120 Protein CON6 gp140CF Protein HIV Isolate Guinea PigNo. Guinea Pig No. Controls (Subtype) 646 647 648 649 GMT 650 651 652653 GMT TriMab₂‡ CD4-IgG2 HIV + Serum SHIV 89.6P*(B) <20 <20 <20 <20 <20<20 <20 <20 <20 <20 NT NT NT SHIV SF162P3*(B) <20 30 48 <20 <20 27 <20<20 <20 <20 NT 0.2 μg/ml NT BX08(B) 270 183 254 55 102 199 64 229 150187 0.7 μg/ml NT 2384 6101(B) <20 38 35 <20 <20 <20 90 72 73 39 1.1μg/ml NT NT BG1168(B) <20 <20 <20 <20 <20 40 <20 <20 25 <20 2.7 μg/ml NTNT 0692(B) 31 32 34 <20 24 28 33 30 45 33 0.8 μg/ml NT  769 PAVO(B) <20<20 <20 <20 <20 <20 <20 <20 <20 <20 2.9 μg/ml NT NT SF162(B) 2,146 308110 282 379 206 5,502 15,098 174 1,313 NT NT >540 SS1196(B) 206 26 14859 83 381 401 333 81 253 NT NT  301# BAL(B) 123 90 107 138 113 107 146136 85 116 NT NT 3307 92RW020(A) <20 <20 <20 <20 <20 <20 <20 <20 <20 <20NT NT  693 DU179(C) <20 43 <20 24 <20 <20 <20 24 515 33 NT 0.8 μg/ml NTDU368(C) 25 35 62 <20 27 <20 <20 <20 23 <20 NT 2.3 μg/ml NT S021(C) <20<20 33 <20 <20 <20 <20 <20 <20 <20 NT 8.3 μg/ml NT S080(C) 24 37 70 4140 <20 <20 <20 52 <20 NT 3.4 μg/ml NT 93ZR001(D) 275 144 126 114 154 306195 129 173 191 NT NT  693 CM244(E) 35 43 64 ND 46 31 25 27 25 26 NT NT 693 *MT-2 Assay; All other HIV isolates were tested in theM7-luciferase assay. HIV-1 isolates QH0692, SS1196, SF162, 6101, BX08,BG1168, BAL were assayed with post-injection 5 serum; other HIV-1isolates were assayed with post-injection 4 serum. ND = not done. HIV +sera was either HIV-1 + human serum (LEH3) or an anti-gp120 guinea pigserum (#) with known neutralizing activity for HIV-1 isolate SS1196. GMT= geometric mean titer of four animals per group. Neutralizing titersreported are after subtraction of any background neutralization inprebleed sera. ‡TriMab₂ = a mixture of human mabs 2F5, b12, 2G12.Conclusions

The production of an artificial HIV-1 Group M consensus env genes(encoding sequences) (CONE and Con-S) have been described that encodes afunctional Env protein that is capable of utilizing the CCR5 co-receptorfor mediating viral entry. Importantly, these Group M consensus envelopegenes could induce T and B cell responses that recognized epitopes ofsubtype B and C HIV-1 primary isolates. In addition, Con-S inducesantibodies that strongly neutralize Subtype-C and A HIV-1 strains (seeTable 3).

The correlates of protection to HIV-1 are not conclusively known.Considerable data from animal models and studies in HIV-1-infectedpatients suggest the goal of HIV-1 vaccine development should be theinduction of broadly-reactive CD4+ and CD8+ anti-HIV-1 T cell responses(Letvin et al, Annu. Rev. Immunol. 20:73-99 (2002)) and high levels ofantibodies that neutralize HIV-1 primary isolates of multiple subtypes(Mascola et al, J. Viral. 73:4009-4018 (1999), Mascola et al, Nat. Med.6:270-210 (2000)).

The high level of genetic variability of HIV-1 has made it difficult todesign immunogens capable of inducing immune responses of sufficientbreadth to be clinically useful. Epitope based vaccines for T and B cellresponses (McMichael et al, Vaccine 20:1918-1921 (2002), Sbai et al,Curr. Drug Targets Infect, Disord. 1:303-313 (2001), Haynes, Lancet348:933-937 (1996)), constrained envelopes reflective of fusionintermediates (Fouts et al, Proc. Natl. Acad. Sci. USA 99:11842-22847(2002)), as well as exposure of conserved high-order structures forinduction of anti-HIV-1 neutralizing antibodies have been proposed toovercome HIV-1 variability (Roben et al, J. Virol. 68:4821-4828 (1994),Saphire et al, Science 293:1155-1159 (2001)). However, with theever-increasing diversity and rapid evolution of HIV-1, the virus is arapidly moving complex target, and the extent of complexity of HIV-1variation makes all of these approaches problematic. The current mostcommon approach to HIV-1 immunogen design is to choose a wild-type fieldHIV-1 isolate that may or may not be from the region in which thevaccine is to be tested. Polyvalent envelope immunogens have beendesigned incorporating multiple envelope immunogens (Bartlett et al,AIDS 12:1291-1300 (1998), Cho et al, J. Virol. 75:2224-2234 (2001)).

The above-described study tests a new strategy for HIV-1 immunogendesign by generating a group M consensus env gene (CON6) with decreasedgenetic distance between this candidate immunogen and wild-type fieldvirus strains. The CON6 env gene was generated for all subtypes bychoosing the most common amino acids at most positions (Gaschen et al,Science 296:2354-2360 (2002), Korber et al, Science 288:1789-1796(2000)). Since only the most common amino acids were used, the majorityof antibody and T cell epitopes were well preserved. Importantly, thegenetic distances between the group M consensus env sequence and anysubtype env sequences was about 15%, which is only half of that betweenwild-type subtypes (30%) (Gaschen et al, Science 296:2354-2360 (2002)).This distance is approximately the same as that among viruses within thesame subtype. Further, the group M consensus env gene was also about 15%divergent from any recombinant viral env gene, as well, since CRFs donot increase the overall genetic divergence among subtypes.

Infectivity of CON6-Env pseudovirions was confirmed using a single-roundinfection system, although the infectivity was compromised, indicatingthe artificial envelope was not in an “optimal” functional conformation,but yet was able to mediate virus entry. That the CON6 envelope usedCCR5 (R5) as its coreceptor is important, since majority of HIV-1infected patients are initially infected with R5 viruses.

BIAcore analysis showed that both CON6 gp120 and gp140CF bound sCD4 anda number of mabs that bind to wild-type HIV-1 Env proteins. Theexpression of the CON6 gp120 and 140CF proteins that are similarantigenically to wild-type HIV-1 envelopes is an important step in HIV-1immunogen development. However, many wild-type envelope proteins expressthe epitopes to which potent neutralizing human mabs bind, yet when usedas immunogens themselves, do not induce broadly neutralizing anti-HIV-1antibodies of the specificity of the neutralizing human mabs.

The neutralizing antibody studies were encouraging in that both CON6gp120, CON6 gp140CF and Con-S gp140CFI induced antibodies thatneutralized select subtype B, C and D HIV-1 primary isolates, with Con-Sgp140CFI inducing the most robust neutralization of non-subtype Bprimary HIV isolates. However, it is clear that the mostdifficult-to-neutralize primary isolates (PAVO, 6101, BG1168, 92RW020,CM244) were either only weakly or not neutralized by anti-CON6 gp120 orgp140 sera (Table 4b). Nonetheless, the Con-S envelope immunogenicityfor induction of neutralizing antibodies is promising, given the breadthof responses generated with the Con-S subunit gp140CFI envelope proteinfor non-subtype B HIV isolates. Previous studies with poxvirusconstructs expressing gp120 and gp160 have not generated high levels ofneutralizing antibodies (Evans et al, J. Infect. Dis. 180:290-298(1999), Polacino et al, J. Virol. 73:618-630 (1999), Ourmanov et al, J.Virol. 74:2960-2965 (2000), Pal et al, J. Virol 76:292-302 (2002),Excler and Plotkin, AIDS 11(Suppl A):S127-137 (1997). rVV expressingsecreted CON6 gp120 and gp140 have been constructed and antibodies thatneutralize HIV-1 primary isolates induced. An HIV neutralizing antibodyimmunogen can be a combination of Con-S gp140CFI, or subunit thereof,with immunogens that neutralize most subtype B isolates.

The structure of an oligomeric gp140 protein is critical when evaluatingprotein immunogenicity. In this regard, study of purified CON6 gp140CFproteins by fast performance liquid chromatography (FPLC) and analyticalultracentrifiguration has demonstrated that the purified gp140 peakconsists predominantly of trimers with a small component of dimers.

Thus, centralized envelopes such as CON6, Con-S or 2003 group M orsubtype consensus or ancestral encoding sequences described herein, areattractive candidates for preparation of various potentially “enhanced”envelope immunogens including CD4-Env complexes, constrained envelopestructures, and trimeric oligomeric forms. The ability of CON6-induced Tand B cell responses to protect against HIV-1 infection and/or diseasein SHIV challenge models will be studied in non-human primates.

The above study has demonstrated that artificial centralized HIV-1 genessuch as group M consensus env gene (CON6) and Con-S can also induce Tcell responses to T cell epitopes in wild-type subtype B and C Envproteins as well as to those on group M consensus Env proteins (FIG. 5).While the DNA prime and rVV boost regimen with CON6 gp140CF immunogenclearly induced IFN-γ producing T cells that recognized subtype B and Cepitopes, further studies are needed to determine if centralizedsequences such as are found in the CON6 envelope are significantlybetter at inducing cross-clade T cell responses than wild-type HIV-1genes (Ferrari et al, Proc. Natl. Acad. Sci. USA 94:1396-1401 (1997),Ferrari et al, AIDS Res. Hum. Retroviruses 16:1433-1443 (2000)).However, the fact that CON6 (and Con-S, env encoding sequence) prime andboosted splenocyte T cells recognized HIV-1 subtype B and C T cellepitopes is an important step in demonstration that CON6 (and Con-S) caninduce T cell responses that might be clinically useful.

Three computer models (consensus, ancestor and center of the tree (COT))have been proposed to generate centralized HIV-1 genes (Gaschen et al,Science 296:2354-2360 (2002), Gao et al, Science 299:1517-1518 (2003),Nickle et al, Science 299:1515-1517 (2003), Korber et al, Science288:1789-1796 (2000). They all tend to locate at the roots of thestar-like phylogenetic trees for most HIV-1 sequences within or betweensubtypes. As experimental vaccines, they all can reduce the geneticdistances between immunogens and field virus strains. However,consensus, ancestral and COT sequences each have advantages anddisadvantages (Gaschen et al, Science 296:2354-2360 (2002), Gao et al,Science 299:1517-1518 (2003), Nickle et al, Science 299:1515-1517(2003). Consensus and COT represent the sequences or epitopes in sampledcurrent wild-type viruses and are less affected by outliers HIV-1sequences, while ancestor represents ancestral sequences that can besignificantly affected by outlier sequences. However, at present, it isnot known which centralized sequence can serve as the best immunogen toelicit broad immune responses against diverse HIV-1 strains, and studiesare in progress to test these different strategies.

Taken together, the data have shown that the HIV-1 artificial CON6 andCon-S envelope can induce T cell responses to wild-type HIV-1 epitopes,and can induce antibodies that neutralize HIV-1 primary isolates, thusdemonstrating the feasibility and promise of using artificialcentralized HIV-1 sequences in HIV-1 vaccine design.

Example 2

HIV-1 Subtype C Ancestral and Consensus Envelope Glycoproteins

Experimental Details

HIV-1 subtype C ancestral and consensus env genes were obtained from theLos Alamos HIV Molecular Immunology Database(http://hiv-web.lanl.gov/immunology), codon-usage optimized formammalian cell expression, and synthesized (FIG. 6). To ensure optimalexpression, a Kozak sequence (GCCGCCGCC) was inserted immediatelyupstream of the initiation codon. In addition to the full-length genes,two truncated env genes were generated by introducing stop codonsimmediately after the gp41 membrane-spanning domain (IVNR) and thegp120/gp41 cleavage site (REKR), generating gp140 and gp120 form of theglycoproteins, respectively (FIG. 8).

Genes were tested for integrity in an in vitro transcription/translationsystem and expressed in mammalian cells. To determine if the ancestraland consensus subtype C envelopes were capable of mediating fusion andentry, gp160 and gp140 genes were co-transfected with an HIV-1/SG3Aenvprovirus and the resulting pseudovirions tested for infectivity usingthe JC53-BL cell assay (FIG. 7). Co-receptor usage and envelopeneutralization sensitivity were also determined with slightmodifications of the JC53-BL assay. Codon-usage optimized andrev-dependent 96ZAM651 env genes were used as contemporary subtype Ccontrols.

Results

Codon-optimized subtype C ancestral and consensus envelope genes (gp160,gp140, gp120) express high levels of env glycoprotein in mammalian cells(FIG. 9).

Codon-optimized subtype C gp160 and gp140 glycoproteins are efficientlyincorporated into virus particles. Western Blot analysis ofsucrose-purified pseudovirions reveals ten-fold higher levels of virionincorporation of the codon-optimized envelopes compared to that of arev-dependent contemporary envelope controls (FIG. 10A).

Virions pseudotyped with either the subtype C consensus gp160 or gp140envelope were more infectious than pseudovirions containing thecorresponding gp160 and gp140 ancestral envelopes. Additionally, gp160envelopes were consistently more infectious than their respective gp140counterparts (FIG. 10B).

Both subtype C ancestral and consensus envelopes utilize CCR5 as aco-receptor to mediate virus entry (FIG. 11).

The infectivity of subtype C ancestral and consensus gp160 containingpseudovirions was neutralized by plasma from subtype C infectedpatients. This suggests that these artificial envelopes possess astructure that is similar to that of native HIV-1 env glycoproteins andthat common neutralization epitopes are conserved. No significantdifferences in neutralization potential were noted between subtype Cancestral and consensus env glycoproteins (gp160) (FIG. 12).

Conclusions

HIV-1 subtype C viruses are among the most prevalent circulatingisolates, representing approximately fifty percent of new infectionsworldwide. Genetic diversity among globally circulating HIV-1 strainsposes a challenge for vaccine design. Although HIV-1 Env protein ishighly variable, it can induce both humoral and cellular immuneresponses in the infected host. By analyzing 70 HIV-1 complete subtype Cenv sequences, consensus and ancestral subtype C env genes have beengenerated. Both sequences are roughly equidistant from contemporarysubtype C strains and thus expected to induce better cross-protectiveimmunity. A reconstructed ancestral or consensus sequencederived-immunogen minimizes the extent of genetic differences betweenthe vaccine candidate and contemporary isolates. However, consensus andancestral subtype C env genes differ by 5% amino acid sequences. Bothconsensus and ancestral sequences have been synthesized for analyses.Codon-optimized subtype C ancestral and consensus envelope genes havebeen constructed and the in vitro biological properties of the expressedglycoproteins determined. Synthetic subtype C consensus and ancestralenv genes express glycoproteins that are similar in their structure,function and antigenicity to contemporary subtype C wild-type envelopeglycoproteins.

Example 3

Codon-Usage Optimization of Consensus of Subtype C gag and nef Genes(C.con.gag and C.con.nef)

Subtype C viruses have become the most prevalent viruses among allsubtypes of Group M viruses in the world. More than 50% of HIV-1infected people are currently carrying HIV-1 subtype C viruses. Inaddition, there is considerable intra-subtype C variability: differentsubtype C viruses can differ by as much as 10%, 6%, 17% and 16% of theirGag, Pol, Env and Nef proteins, respectively. Most importantly, thesubtype C viruses from one country can vary as much as the virusesisolated from other parts of the world. The only exceptions are HIV-1strains from India/China, Brazil and Ethiopia/Djibouti where subtype Cappears to have been introduced more recently. Due to the high geneticvariability of subtype C viruses even within a single country, animmunogen based on a so single virus isolate may not elicit protectiveimmunity against other isolates circulating in the same area.

Thus gag and nef gene sequences of subtype C viruses were gathered togenerate consensus sequences for both genes by using a 50% consensusthreshold. To avoid a potential bias toward founder viruses, only onesequence was used from India/China, Brazil and Ethiopia/Djibouti,respectively, to generate the subtype C consensus sequences (C.con.gagand C.con.nef). The codons of both C.con.gag and C.con.nef genes wereoptimized based on the codon usage of highly expressed human genes. Theprotein expression following transfection into 293T cells is shown inFIG. 13. As can be seen, both consensus subtype C Gag and Nef proteinswere expressed efficiently and recognized by Gag- and Nef-specificantibodies. The protein expression levels of both C.con.gag andC.con.nef genes are comparible to that of native subtype env gene(96ZM651).

Example 4

Synthesis of a Full Length “Consensus of the Consensus env Gene withConsensus Variable Regions” (CON-S)

In the synthesized “consensus of the consensus” env gene (CON6), thevariable regions were replaced with the corresponding regions from acontemporary subtype C virus (98CN006). A further con/con gene has beendesigned that also has consensus variable regions (CON-s). The codons,of the Con-S env gene were optimized based on the codon usage of highlyexpressed human genes. (See FIGS. 14A and 14B for amino acid sequencesand nucleic acid sequences, respectfully.)

Paired oligonucleotides (80-mers) which overlap by 20 bp at their 3′ends and contain invariant sequences at their 5′ and 3′ ends, includingthe restriction enzyme sites EcoRI and BbsI as well as BsmBI and BamHI,respectively, were designed. BbsI and BamHI are Type II restrictionenzymes that cleave outside of their recognition sequences. They havebeen positioned in the oligomers in such a way that they cleave thefirst four resides adjacent to the 18 bp invariant region, leaving 4base 5′ overhangs at the end of each fragment for the following ligationstep. 26 paired oligomers were linked individually using PCR and primerscomplimentary to the 18 bp invariant sequences. Each pair was clonedinto pGEM-T (Promega) using the T/A cloning method and sequenced toconfirm the absence of inadvertent mutations/deletions. pGEM-T subclonescontaining the proper inserts were then digested, run on a 1% agarosegel, and gel purified (Qiagen). Four individual 108-mers were ligatedinto pcDNA3.1 (Invitrogen) in a multi-fragment ligation reaction. Thefour-way ligations occurred among groups of fragments in a stepwisemanner from the 5′ to the 3′ end of the gene. This process was repeateduntil the entire gene was reconstructed in the pcDNA3.1 vector.

A complete Con-S gene was constructed by ligating the codon usageoptimized oligo pairs together. To confirm its open reading frame, an invitro transcription and translation assay was performed. Proteinproducts were labeled by S³⁵-methionine during the translation step,separated on a 10% SDS-PAGE, and detected by radioautography. Expectedsize of the expressed Con-S gp160 was identified in 4 out of 7 clones(FIG. 14C).

CONs Env protein expression in the mammalian cells after transfectedinto 293T cells using a Western blot assay (FIG. 15). The expressionlevel of Con-S Env protein is very similar to what was observed from theprevious CON6 env clone that contains the consensus conservative regionsand variable loops from 98CN006 virus isolate.

The Env-pseudovirons was produced by cotransfecting Con-S env clone andenv-deficient SG3 proviral clone into 293T cells. Two days aftertransfection, the pseudovirions were harvested and infected intoJC53BL-13 cells. The infectious units (IU) were determined by countingthe blue cells after staining with X-gal in three independentexperiments. When compared with CON6 env clone, Con-S env clones producesimilar number of IU in JC53BL-13 cells (FIG. 16). The IU titers forboth are about 3 log higher than the SG3 backbone clone control (NoEnv). However, the titers are also about 2 log lower than the positivecontrol (the native HIV-1 env gene, NL4-3 or YU2). These data suggestthat both consensus group M env clones are biologically functional.Their functionality, however, has been compromised. The functionalconsensus env genes indicate that these Env proteins fold correctly,preserve the basic conformation of the native Env proteins, and are ableto be developed as universal Env immunogens.

It was next determined what coreceptor Con-S Env uses for its entry intoJC53-BL cells. When treated with CXCR4 blocking agent AMD3100, theinfectivity of NL4-3 Env-pseudovirons was blocked while the infectivityof YU2, Con-S or CON6 Env-pseudovirons was not inhibited. In contrast,when treated with CCR5 blocking agent TAK779, the infectivity of NL4-3Env-pseudovirons was not affected, while the infectivity of YU2, Con-Sor CON6 Env-pseudovirons was inhibited. When treated with both blockingagents, the infectivity of all pseudovirions was inhibited. Takentogether, these data show that the Con-S as well as CON6 envelope usesthe CCR5 but not CXCR4 co-receptor for its entry into target cells.

It was next determined whether CON6 or Con-S Env proteins could beequally efficiently incorporated in to the pseudovirions. To be ableprecisely compare how much Env proteins were incorporated into thepseudovirions, each pseudovirions is loaded on SDS-PAGE at the sameconcentraion: 5 μg total protein for cell lysate, 25 ng p24 for cellculture supernatant, or 150 ng p24 for purified virus stock(concentrated pseudovirions after super-speed centrifugation). There wasno difference in amounts of Env proteins incorporated in CON6 or Con-SEnv-pseudovirions in any preparations (cell lysate, cell culturesupernatant or purified virus stock) (FIG. 17).

Example 5

Synthesis of a Consensus Subtype A Full Length env (A.con.env) Gene

Subtype A viruses are the second most prevalent HIV-1 in the Africancontinent where over 70% of HIV-1 infections have been documented.Consensus gag, env and nef genes for subtype C viruses that are the mostprevalent viruses in Africa and in the world were previously generated.Since genetic distances between subtype A and C viruses are as high as30% in the env gene, the cross reactivity or protection between bothsubtypes will not be optimal. Two group M consensus env genes for allsubtypes were also generated. However, to target any particular subtypeviruses, the subtype specific consensus genes will be more effectivesince the genetic distances between subtype consensus genes and fieldviruses from the same subtype will be smaller than that between group Mconsensus genes and these same viruses. Therefore, consensus genes needto be generated for development of subtype A specific immunogens. Thecodons of the A.con.env gene were optimized based on the codon usage ofhighly expressed human genes. (See FIGS. 18A and 18B for amino acid andnucleic acid sequences, respectively.)

Each pair of the oligos has been amplified, cloned, ligated andsequenced. After the open reading frame of the A.con env gene wasconfirmed by an in vitro transcription and translation system, the A.conenv gene was transfected into the 293T cells and the protein expressionand specificity confirmed with the Western blot assay (FIG. 18). It wasthen determined whether A.con envelope is biologically functional. Itwas co-transfected with the env-defective SG3 proviral clone into 293Tcells. The pseudotyped viruses were harvested and used to infect JC53BLcells. Blue cells were detected in JC53-BL cells infected with the A.conEnv-pseudovirions, suggesting that A.con Env protein is biologicallyfunctional (Table 6). However, the infectious titer of A.conEnv-psuedovirions was about 7-fold lower than that of pseudovirions withwild-type subtype C envelope (Table 6). Taken together, the biologicalfunction A.con Env proteins suggests that it folds correctly and mayinduce linear and conformational T and B cell epitopes if used as an Envimmunogen.

TABLE 6 Infectivity of pseudovirons with A.con env genes JC53BL13(IU/ul) Mar. 31, 2003 Apr. 7, 2003 Apr. 25, 2003 non filtered supt. 0.22μm filtered 0.22 μm filtered A.con + SG3 4 8.5 15.3 96ZM651 + SG3 87 133104 SG3 backbone 0 0.07 0.03 Neg control 0 0.007 0

Example 6

Design of Full Length “Consensus of the Consensus gag, pol and nefGenes” (M.con.gag, M.con.pol and M.con.nef) and a Subtype C Consensuspol Gene (C.con.pol)

For the group M consensus genes, two different env genes wereconstructed, one with virus specific variable regions (CON6) and onewith consensus variable regions (Con-S). However, analysis of T cellimmune responses in immunized or vaccinated animals and humans showsthat the env gene normally is not a main target for T cell immuneresponse although it is the only gene that will induce neutralizingantibody. Instead, HIV-1 Gag, Pol and Nef proteins are found to beimportant for inducing potent T cell immune responses. To generate arepertoire of immunogens that can induce both broader humoral andcellular immune responses for all subtypes, it may be necessary toconstruct other group M consensus genes other than env gene alone.“Consensus of the consensus” gag, pol and nef genes (M.con.gag.,M.con.pol and M.con.nef) have been designed. To generate a subtypeconsensus pol gene, the subtype C consensus poi gene (C.con.pol) wasalso designed. The codons of the M.con.gag., M.con.pol, M.con.nef andC.con.pol. genes were optimized based on the codon usage of highlyexpressed human genes. (See FIG. 19 for nucleic acid and amino acidsequences.)

Example 7

Synthetic Subtype B Consensus gag and env Genes

Experimental Details

Subtype B consensus gag and env sequences were derived from 37 and 137contemporary HIV-1 strains, respectively, codon-usage optimized formammalian cell expression, and synthesized (FIGS. 20A and 20B). Toensure optimal expression, a Kozak sequence (GCCGCCGCC) was insertedimmediately upstream of the initiation codon. In addition to thefull-length env gene, a truncated env gene was generated by introducinga stop codon immediately after the gp41 membrane-spanning domain (IVNR)to create a gp145 gene. Genes were tested for integrity in an in vitrotranscription/translation system and expressed in mammalian cells.(Subtype B consensus Gag and Env sequences are set forth in FIGS. 20Cand 20D, respectively.)

To determine if the subtype B consensus envelopes were capable ofmediating fusion and entry, gp160 and gp145 genes were co-transfectedwith an HIV-1/SG3Δenv provirus and the resulting pseudovirions weretested for infectivity using the JC53-BL cell assay. JC53-BL cells are aderivative of HeLa cells that express high levels of CD4 and the HIV-1coreceptors CCR5 and CXCR4. They also contain the reporter cassettes ofluciferase and β-galactosidase that are each expressed from an HIV-1LTR. Expression of the reporter genes is dependent on production ofHIV-1 Tat. Briefly, cells are seeded into 24-well plates, incubated at37° C. for 24 hours and treated with DEAE-Dextran at 37° C. for 30 min.Virus is serially diluted in 1% DMEM, added to the cells incubating inDEAE-dextran, and allowed to incubate for 3 hours at 37° C. after whichan additional 500 μL of cell media is added to each well. Following afinal 48-hour incubation at 37° C., cells are fixed, stained usingX-Gal, and overlaid with PBS for microscopic counting of blue foci.Counts for mock-infected wells, used to determine background, aresubtracted from counts for the sample wells. Co-receptor usage andenvelope neutralization sensitivity were also determined with slightmodifications of the JC53-BL assay.

To determine whether the subtype B consensus Gag protein was capable ofproducing virus-like particles (VLPs) that incorporated Envglycoproteins, 293T cells were co-transfected with subtype B consensusgag and env genes. 48-hours post-transfection, cell supernatantscontaining VLPs were collected, clarified in a tabletop centrifuge,filtered through a 0.2 mM filter, and pellet through a 20% sucrosecushion. The VLP pellet was resuspended in PBS and transferred onto a20-60% continuous sucrose gradient. Following overnight centrifugationat 100,000×g, 0.5 ml fractions were collected and assayed for p24content. The refractive index of each fraction was also measured.Fractions with the correct density for VLPs and containing the highestlevels of p24 were pooled and pellet a final time. VLP-containingpellets were re-suspended in PBS and loaded on a 4-20% SDS-PAGE gel.Proteins were transferred to a PVDF membrane and probed with serum froma subtype B HIV-1 infected individual.

Results

Codon-usage optimized, subtype B consensus envelope (gp160, gp145) andgag genes express high levels of glycoprotein in mammalian cells (FIG.21).

Subtype B gp160 and gp145 glycoproteins are efficiently incorporatedinto virus particles. Western Blot analysis of sucrose-purifiedpseudovirions suggests at least five-fold higher levels of consensus Benvelope incorporation compared to incorporation of a rev-dependentcontemporary envelope (FIG. 23A). Virions pseudotyped with either thesubtype B consensus gp160 or gp145 envelope are more infectious thanpseudovirions containing a rev-dependent contemporary envelope (FIG. 23B).

Subtype B consensus envelopes utilize CCR5 as the co-receptor to gainentry into CD4 bearing target cells (FIG. 22).

The infectivity of pseudovirions containing the subtype B consensusgp160 envelope was neutralized by plasma from HIV-1 subtype B infectedpatients (FIG. 24C) and neutralizing monoclonal antibodies (FIG. 24A).This suggests that the subtype B synthetic consensus B envelopes issimilar to native HIV-1 Env glycoproteins in its overall structure andthat common neutralization epitopes remain intact. FIGS. 24B and 24Dshow neutralization profiles of a subtype B control envelope (NL4.3Env).

Subtype B consensus Gag proteins are able to bud from the cell membraneand form virus-like particles (FIG. 25A). Co-transfection of thecodon-optimized subtype B consensus gag and gp160 genes produces VLPswith incorporated envelope (FIG. 25B).

Conclusions

The synthetic subtype B consensus env and gag genes express viralproteins that are similar in their structure, function and antigenicityto contemporary subtype B Env and Gag proteins. It is contemplated thatimmunogens based on subtype B consensus genes will elicit CTL andneutralizing immune responses that are protective against a broad set ofHIV-1 isolates.

All documents and other information sources cited above are herebyincorporated in their entirety by reference. Also incorporated byreference is Liao et al, J. Virol. 78:5270 (2004)).

What is claimed is:
 1. A nucleic acid comprising a nucleotide sequencethat encodes a group M consensus Env comprising SEQ ID NO: 13, or aCons-S gp140CFI Env comprising SEQ ID NO: 30, or a CON-S gp140CF Envcomprising SEQ ID NO:
 36. 2. The nucleic acid according to claim 1,wherein said nucleotide sequence comprises codons optimized forexpression in human cells.
 3. The nucleic acid according to claim 2,wherein said nucleic acid comprises the nucleotide sequence set forth inSEQ ID NO:
 14. 4. The nucleic acid according to claim 2, wherein saidnucleic acid comprises the nucleotide sequence set forth in SEQ ID NO:31.
 5. The nucleic acid according to claim 2, wherein said nucleic acidcomprises the nucleotide sequence set forth in SEQ ID NO:
 37. 6. Anucleic acid comprising a nucleotide sequence that encodes a recombinantgp120 envelope protein, wherein the recombinant gp120 envelope proteincomprises the consecutive amino acid sequence represented by amino acidnumbers 30 to 503 of SEQ ID NO:
 13. 7. The nucleic acid of claim 6comprising the consecutive nucleotide sequence from SEQ ID NO: 14 thatencodes the consecutive amino acid sequence represented by amino acidnumbers 30 to 503 of SEQ ID NO: 13, wherein said nucleotide sequencecomprises codons optimized for expression in human cells.
 8. A vectorcomprising the nucleic acid according to claim
 1. 9. An isolatedmammalian cell comprising a nucleic acid according to claim 1 forrecombinant protein expression.
 10. A vector comprising the nucleicacids of claim
 6. 11. A composition comprising the nucleic acidaccording to claim 1 and a carrier.
 12. A method of inducing an immuneresponse in a mammal, the method comprising administering to said mammalthe composition of claim 11 in an amount sufficient to effect suchinduction.
 13. A composition comprising the nucleic acid according toclaim 1 and a carrier, wherein the nucleic acid comprises a nucleotidesequence that encodes a group M consensus Env comprising SEQ ID NO: 13and a carrier.
 14. A composition comprising the nucleic acid accordingto claim 6 and a carrier.
 15. An isolated mammalian cell comprising thenucleic acid according to claim 1, wherein the nucleic acid comprises anucleotide sequence that encodes a group M consensus Env comprising SEQID NO: 13 or recombinant protein expression.
 16. An isolated mammaliancell comprising the nucleic acid according to claim 6 for recombinantprotein expression.
 17. A method of inducing an immune response in amammal, the method comprising administering to said mammal thecomposition of claim 13 in an amount sufficient to effect suchinduction.
 18. A method of inducing an immune response in a mammal, themethod comprising administering to said mammal the composition of claim14 in an amount sufficient to effect such induction.